U.S. patent application number 15/101583 was filed with the patent office on 2016-10-27 for thymic stromal lymphopoietin receptor-specific chimeric antigen receptors and methods using same.
This patent application is currently assigned to The United States of America, as represented by the Secretary, Dept. of Health and Human Services. The applicant listed for this patent is The United States of America, as represented by the Secretary, Dept. of Health and Human Services, The United States of America, as represented by the Secretary, Dept. of Health and Human Services. Invention is credited to Terry J. Fry, Haiying Qin.
Application Number | 20160311910 15/101583 |
Document ID | / |
Family ID | 51946020 |
Filed Date | 2016-10-27 |
United States Patent
Application |
20160311910 |
Kind Code |
A1 |
Qin; Haiying ; et
al. |
October 27, 2016 |
THYMIC STROMAL LYMPHOPOIETIN RECEPTOR-SPECIFIC CHIMERIC ANTIGEN
RECEPTORS AND METHODS USING SAME
Abstract
The invention provides a chimeric antigen receptor (CAR)
comprising an antigen binding domain specific for TSLPR, a
transmembrane domain, and an intracellular T cell signaling domain.
Nucleic acids, recombinant expression vectors, host cells,
populations of cells, antibodies, or antigen binding portions
thereof, and pharmaceutical compositions relating to the CARs are
disclosed. Methods of detecting the presence of a proliferative
disorder, e.g., cancer, in a mammal and methods of treating or
preventing a proliferative disorder, e.g., cancer, in a mammal are
also disclosed.
Inventors: |
Qin; Haiying; (Potomac,
MD) ; Fry; Terry J.; (Bethesda, MD) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
The United States of America, as represented by the Secretary,
Dept. of Health and Human Services |
Bethesda |
MD |
US |
|
|
Assignee: |
The United States of America, as
represented by the Secretary, Dept. of Health and Human
Services
Bethesda
MD
|
Family ID: |
51946020 |
Appl. No.: |
15/101583 |
Filed: |
October 30, 2014 |
PCT Filed: |
October 30, 2014 |
PCT NO: |
PCT/US2014/063096 |
371 Date: |
June 3, 2016 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
61991697 |
May 12, 2014 |
|
|
|
61912948 |
Dec 6, 2013 |
|
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Current U.S.
Class: |
1/1 |
Current CPC
Class: |
C07K 2319/03 20130101;
C07K 2319/02 20130101; C07K 2317/524 20130101; C07K 14/70517
20130101; C07K 16/2803 20130101; C07K 14/7051 20130101; C07K
2319/00 20130101; C07K 2317/565 20130101; A61P 35/00 20180101; C07K
2317/526 20130101; A61P 35/02 20180101; C07K 16/3061 20130101; C07K
2319/33 20130101; C07K 16/2866 20130101; C07K 2317/622 20130101;
C07K 14/7151 20130101 |
International
Class: |
C07K 16/28 20060101
C07K016/28; C07K 14/725 20060101 C07K014/725; C07K 14/715 20060101
C07K014/715; C07K 16/30 20060101 C07K016/30; C07K 14/705 20060101
C07K014/705 |
Claims
1. A chimeric antigen receptor (CAR) comprising an antigen binding
domain specific for TSLPR, a transmembrane domain, and an
intracellular T cell signaling domain.
2. The CAR according to claim 1, wherein the antigen binding domain
comprises the light chain variable region comprising the sequences
of SEQ ID NOS: 18, 20, 22, 24, 25, 27, and 29 or SEQ ID NOS: 19,
21, 23, 24, 26, 28, and 29.
3. The CAR according to claim 1, wherein the antigen binding domain
comprises the heavy chain variable region comprising the sequences
of SEQ ID NOS: 6, 7, 9, 10, 11, 13, and 15 or SEQ ID NOS; 6, 8, 9,
10, 12, 14, and 16.
4. The CAR according to claim 1, wherein the antigen binding domain
comprises the linker sequence of SEQ ID NO: 17.
5. The CAR according to claim 1, wherein the antigen binding domain
comprises SEQ ID NO: 1.
6. (canceled)
7. The CAR according to claim 1, wherein the transmembrane domain
comprises CD8 amino acid sequence comprising the CD8.alpha. hinge
sequence of SEQ ID NO: 35 and the transmembrane domain of sequence
SEQ ID NO: 36.
8. (canceled)
9. The CAR according to claim 1, wherein the intracellular T cell
signaling domain comprises the 4-1BB amino acid sequence of SEQ ID
NO: 37.
10. The CAR according to claim 1, wherein the intracellular T cell
signaling domain comprises the CD3 zeta amino acid sequence of SEQ
ID NO: 38.
11. The CAR according to claim 1, wherein the CAR further comprises
the spacer comprising SEQ ID NOS: 30-33.
12. The CAR according to claim 1, wherein the CAR comprises any one
of the sequences of SEQ ID NO: 39-46.
13. A nucleic acid comprising a nucleotide sequence encoding the
CAR according to claim 1.
14. (canceled)
15. A recombinant expression vector comprising the nucleic acid
according to claim 13.
16. The recombinant expression vector according to claim 15,
wherein the recombinant expression vector is a lentiviral
vector.
17. An isolated host cell comprising the recombinant expression
vector of claim 15.
18.-19. (canceled)
20. A pharmaceutical composition comprising the CAR of claim 1, and
a pharmaceutically acceptable carrier.
21. A method of detecting the presence of cancer, comprising: (a)
contacting a sample comprising one or more cells with the CAR of
claim 1, thereby forming a complex, and (b) detecting the complex,
wherein detection of the complex is indicative of the presence of
cancer.
22. The method of claim 21, wherein the cancer is BCP-ALL.
23.-25. (canceled)
26. A method of determining whether a subject with a proliferative
disorder is a candidate for treatment with a chimeric antigen
receptor comprising an antigen binding domain specific for TSLPR,
the method comprising: measuring TSLPR expression levels in a
biological sample from the subject; and determining if the TSLPR
expression levels of the biological sample are increased compared
to a sample from a control subject without the proliferative
disorder.
27. A method of treating or preventing cancer in a mammal, the
method comprising administering to the mammal an effective amount
of the CAR of claim 1 to treat or prevent the cancer in the
mammal.
28. The method of claim 27, wherein the cancer is BCP-ALL.
29. A method of treating or preventing a proliferative disorder in
a mammal, the method comprising administering to the mammal an
effective amount of the CAR of claim 1 to treat or prevent the
proliferative disorder in the mammal, wherein the proliferative
disorder is associated with a mutation in the IKZF gene.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This patent application claims the benefit of U.S.
Provisional Patent Application No. 61/912,948, filed Dec. 6, 2013
and U.S. Provisional Patent Application No. 61/991,697, filed May
12, 2014, each of which is incorporated herein by reference in its
entirety.
INCORPORATION-BY-REFERENCE OF MATERIAL SUBMITTED ELECTRONICALLY
[0002] Incorporated by reference in its entirety herein is a
computer-readable nucleotide/amino acid sequence listing submitted
concurrently herewith and identified as follows: One 58,011 Byte
ASCII (Text) file named "718601ST25," created on Sep. 11, 2014.
BACKGROUND OF THE INVENTION
[0003] Cancer is a public health concern. Despite advances in
treatments such as chemotherapy, the prognosis for many cancers
continues to be poor. Accordingly, there exists an unmet need for
additional treatments for cancer.
BRIEF SUMMARY OF THE INVENTION
[0004] The invention provides chimeric antigen receptors (CARs)
comprising an antigen binding domain specific for thymic stromal
lymphopoietin receptor (TSLPR), a transmembrane domain, and an
intracellular T cell signaling domain. The CAR may further comprise
a 4-1BB intracellular domain, a spacer, or both.
[0005] Further embodiments of the invention provide related nucleic
acids, recombinant expression vectors, host cells, populations of
cells, antibodies, or antigen binding portions thereof, and
pharmaceutical compositions relating to the CARs of the
invention.
[0006] Additional embodiments of the invention provide methods of
detecting the presence of a proliferative disorder, e.g., cancer,
and methods of treating or preventing a proliferative disorder,
e.g., cancer, in a mammal.
BRIEF DESCRIPTION OF THE DRAWINGS
[0007] FIG. 1 presents flow cytometry graphs showing the binding of
a 3G11 anti-TSLPR antibody and a commercially available anti-TSLPR
antibody to precursor-B cell acute lymphoblastic leukemias
overexpressing TSLPR. The y-axis is counts, x-axis is mean
fluorescence intensity. Binding was detected using
phycoerytherin-conjugated goat anti-mouse antibody.
[0008] FIG. 2 presents flow cytometry graphs showing the surface
expression of CD19, CD22, and TSLPR on stable leukemia cell lines
and JH331, which is derived from patient leukemia blast, naturally
overexpressed TSLPR, and cannot be cultured in vitro. The y-axis is
counts, x-axis is mean fluorescence intensity.
[0009] FIG. 3 presents diagrammatic representations of short and
long CARs in accordance with certain embodiments of the present
invention.
[0010] FIG. 4 presents a diagrammatic representation of
construction of a vector encoding a CAR in accordance with certain
embodiments of the present invention.
[0011] FIGS. 5A and 5B show flow cytometry graphs showing
transduction of human T cells using CARs in accordance with certain
embodiments of the present invention. FIG. 5A shows detection of
CAR using Protein L. FIG. 5B shows detection of CAR using a CD22
protein Fc construct.
[0012] FIG. 6 is a bar graph showing cytolytic cytokine release by
TSLPR CAR transduced T cells in accordance with certain embodiments
of the present invention.
[0013] FIGS. 7A-7H and 8A-8E are bar graphs showing T cells with
TSLPR CAR produce a broad range of inflammatory cytokines in the
presence of both TSLPR-transduced and naturally overexpressing ALL
cells in accordance with certain embodiments of the present
invention.
[0014] FIGS. 9A-D are line graphs showing TSLPR CAR mediated tumor
cell lysis in accordance with certain embodiments of the present
invention.
[0015] FIG. 10 shows images of leukemia reduction in vivo with
short TSLPR CARs in accordance with certain embodiments of the
present invention. Day 0: 5E5 cells REH-TSLPR, Day 4: 15E6 cells
CAR T. The fourth animal in the short CAR column at Day 23 and Day
30 is not shown since the animal died due to a wasting syndrome
consistent with xenographic graph versus host disease.
[0016] FIG. 11 is a dot plot showing comparison of the ALL in blood
with the treatment of T cells transduced with different types of
constructs in accordance with certain embodiments of the present
invention. Peripheral ALL Burden on day 27 Post ADT (adoptive
transfer).
[0017] FIG. 12A is a dot plot showing percentage and persistence of
CAR T cells in vivo post adoptive transfer in accordance with
certain embodiments of the present invention. p=0.0008 for short
CAR day 27 and long CAR days 16 and 27. There is evidence for
increased numbers of short CAR T cells on day 16, although this is
not significant.
[0018] FIG. 12B is a dot plot of flow analysis displaying a typical
quantity of CAR T cells in blood in accordance with certain
embodiments of the present invention.
[0019] FIG. 13 presents images showing that reduction of leukemia
in vivo with TSLPR CAR is target specific in accordance with
certain embodiments of the present invention. On day 0: 1E6 tumor
cells; on day 16: treated with 10E6 cells short CAR.
[0020] FIG. 14 is a bar graph showing TSLPR CAR transduced T cells
are skewed to CD8 post ADT (on day 50) in accordance with certain
embodiments of the present invention. The slightly increased
relative number of CD4+ CAR T cells following CD3/CD28
bead-mediated expansion prior to infusion converts to a
predominance of CD8+/TSLPR CAR+ (measured by TSLPR Fc) at day 50
following injection.
[0021] FIG. 15 is a dot plot showing the physical distribution of
the TSLPR CAR in accordance with certain embodiments of the present
invention. CD45RA+CCR7+ are naive, CD45RA-CCR7+ are central memory,
and CD45RA+CCR7- are effector memory phenotypes of the T cells.
[0022] FIG. 16A presents bioluminescent images tracking leukemia
progression with different treatments in vivo in accordance with
certain embodiments of the present invention.
[0023] FIG. 16B is a line graph showing quantitation of leukemia
progression in accordance with certain embodiments of the present
invention.
[0024] FIG. 16C is a line graph showing a survival plot of TSLPR
CAR treatment in accordance with certain embodiments of the present
invention.
[0025] FIG. 17 presents images showing reduction of high burden in
patient TSLPRhi xenografts using TSLPR short CAR in accordance with
certain embodiments of the present invention.
[0026] FIG. 18A is a dot plot showing analysis in blood of patient
TSLPRhi xenografts 22 days post tumor challenge in accordance with
certain embodiments of the present invention. 1E6 cells of JH352 or
NH362 treated with 15E6 cells TSLPR-short CAR T cell or Lenti-GFP T
cell in NSG mice.
[0027] FIG. 18B is a dot plot showing analysis in bone marrow of
patient TSLPRhi xenografts 22 days post tumor challenge in
accordance with certain embodiments of the present invention. 1E6
of JH352 or NH362 treated with 15E6 TSLPR-short CAR T cell or
Lenti-GFP T cell in NSG mice.
[0028] FIG. 19 presents images showing treatment of Patient
JH331-Luc with 1.2E6 Short TSLPR CAR where the short TSLPR CAR can
reduce ALL in patient xerographs with as low as 1.2 million of CAR
T cells in accordance with certain embodiments of the present
invention.
[0029] FIG. 20 is a dot plot showing the percentage of CAR T cells
presented in mouse blood sample in accordance with certain
embodiments of the present invention.
[0030] FIG. 21 is a bar graph showing the shift of the CD4 to CD8
of CART cells after injection in vivo in accordance with certain
embodiments of the present invention.
[0031] FIG. 22 is a representative dot plot on day 35 following
leukemia injection in accordance with certain embodiments of the
present invention.
[0032] FIG. 23 is a line plot showing survival following injection
of aggressive TSLPRhi ALL into NSG mice with and without TSLPR CAT
treatment (2.times.10.sup.6/mouse) on day 18 (n=5/group) in
accordance with certain embodiments of the present invention.
[0033] FIG. 24 presents images showing results based on CAR+ T
cells (3E6) of TSLPR, CD19, and CD22 injected into NSG mice
engrafted with the patient xenograph cell line JH331-LUC for 29
days, in accordance with certain embodiments of the present
invention. T cells transduced with GFP were used as a negative
control.
[0034] FIGS. 25A-C are line graphs showing percent lysis of tumor
cells using CARs in accordance with certain embodiments of the
present invention.
[0035] FIG. 26 presents images showing therapeutic function of the
different CAR constructs in vivo in accordance with certain
embodiments of the present invention.
DETAILED DESCRIPTION OF THE INVENTION
[0036] Acute lymphoblastic leukemia (ALL) represents a common
oncologic diagnosis in children. Substantial progress has been made
in the upfront chemotherapy for pediatric ALL such that most of
patients will be cured. Nonetheless, ALL remains a common cause of
death from cancer in children due to relapse of disease that no
longer responds to cytotoxic chemotherapy or due to refractoriness
to upfront treatment. Furthermore, long-term therapy-induced
morbidity remains a major issue, particularly in those patients
deemed to be high-risk for relapse and thus treated with more
intense regimens under current risk-adapted protocols. In adults,
ALL occurs less commonly than in children but the prognosis for
adult ALL is worse than in children undergoing standard cytotoxic
chemotherapy. Treatment of young adults on pediatric-type regimens
has improved outcome but not to the level achieved in children.
[0037] The adoptive cell transfer (ADT or ACT) of T cells
genetically modified to express chimeric antigen receptors (CARs)
targeting antigens expressed on lymphoid cells have demonstrated
potent activity in B cell malignancies including ALL resulting in
remissions in chemotherapy refractory patients. The surface protein
being targeted in the majority of these trials is the CD19 antigen
that is expressed on both malignant and non-malignant B cells.
However, not all patients respond and relapses occur, in some cases
due to loss of CD19 expression. Loss of CD19 also has been observed
after treatment with bispecific antibody-based reagents targeting
CD19 and CD3.
[0038] Substantial progress in genomics has resulted in the
identification of genes and pathways that are dysregulated in ALL.
One such category are those associated with cytokine signaling
including IL-7 and, in particular, CD127 (IL-7Ralpha). Thymic
stromal lymphopoietin (TSLP) is a cytokine that shares CD127 but
utilizes a second receptor chain, TSLPR (gene name CRLF2) as part
of the heterodimeric signaling complex. Overexpression of TSLPR has
been identified in 5-10% of pediatric and adult ALL, largely due to
translocations or deletions resulting in alternative promoters.
Overexpression of TSLPR appears to be associated with poor
prognosis in both children and adults with ALL, and it appears that
activation of the TSLPR pathway as biologically important for ALL
blasts. Also, in approximately 50% of cases, increased TSLPR
expression is associated with mutations in the IKZF gene, a
particularly high risk subgroup of patients. TSLPR seems to have
restricted normal tissue expression.
[0039] An embodiment of the invention provides chimeric antigen
receptors (CARs) comprising an antigen binding domain specific for
TSLPR, a transmembrane domain, and an intracellular T cell
signaling domain. The CAR may further comprise a 4-1BB
intracellular domain, a spacer, or both.
[0040] A chimeric antigen receptor (CAR) is an artificially
constructed hybrid protein or polypeptide containing the antigen
binding domain of an antibody (e.g., single chain variable fragment
(scFv)) linked to T-cell signaling domains. Characteristics of CARs
include their ability to redirect T-cell specificity and reactivity
toward a selected target in a non-MHC-restricted manner, exploiting
the antigen-binding properties of monoclonal antibodies. The
non-MHC-restricted antigen recognition gives T cells expressing
CARs the ability to recognize antigen independent of antigen
processing, thus bypassing a major mechanism of tumor escape.
Moreover, when expressed in T-cells, CARs advantageously do not
dimerize with endogenous T cell receptor (TCR) alpha and beta
chains.
[0041] The phrases "have antigen specificity" and "elicit
antigen-specific response" as used herein means that the CAR can
specifically bind to and immunologically recognize an antigen, such
that binding of the CAR to the antigen elicits an immune
response.
[0042] The CARs of the invention have antigen specificity for
Thymic Stromal Lymphopoietin Receptor (TSLPR). TSLPR is
overexpressed on the surface of approximately 10% of adult and
pediatric B cell precursor acute lymphoblastic leukemia (BCP-ALL).
The expression of TSLPR by normal, non-tumor, or non-cancerous
cells is not as robust as the expression by tumor or cancer cells.
In this regard, the tumor or cancer cells can overexpress TSLPR or
express TSLPR at a significantly higher level, as compared to the
expression of TSLPR by normal, non-tumor, or non-cancerous
cells.
[0043] Without being bound to a particular theory or mechanism, it
is believed that by eliciting an antigen-specific response against
TSLPR, the inventive CARs provide for one or more of the following:
targeting and destroying TSLPR-expressing cancer cells, reducing or
eliminating cancer cells, facilitating infiltration of immune cells
to tumor site(s), and enhancing/extending anti-cancer
responses.
[0044] The invention provides a CAR comprising an antigen binding
domain specific for TSLPR, based on the antibodies, e.g., 3G11 as
described in Lu et al., J. Exp. Med., 2009, 206:2111-9 or 2D10 as
described in Rochman et al., J. Immunol., 2007, 178:6720-6724 (each
incorporated herein by reference in its entirety). The scFv of
these antibodies comprise a light chain variable region and a heavy
chain variable region. In embodiments of the invention, the light
chain and heavy chain may comprise any suitable combination of
light chain and heavy chain sequences, e.g., as listed in Table 1
below.
[0045] In an embodiment, the antigen binding domain comprises a
linker. The linker connects the heavy chain variable region and the
light chain variable region of the antigen binding domain. Any
linker suitable for linking the heavy chain variable region and the
light chain variable region may be used in the antigen binding
domains of the invention. In an embodiment, the linker comprises,
consists of, or consists essentially of a glycine-serine linker
domain. Preferably, the antigen binding domain comprises a scFv
comprising a heavy chain variable region, a light chain variable
region, and a linker. In embodiments of the invention, the light
chain, heavy chain, and linker may comprise any suitable
combination of light chain, heavy chain, and linker sequences as
listed in Table 1 below.
[0046] In an embodiment of the invention, the antigen binding
domain that comprises an scFv comprising, consisting, or consisting
essentially of
TABLE-US-00001 (SEQ ID NO: 1)
QVTLKESGPGILKPSQTLSLTCSFSGFSLSTSGMGVGWIRQPSGKGLEWL
AHIWWDDDKYYNPSLKSQLTISKDTSRNQVFLKITSVDTADTATYYCSRR
PRGTMDAMDYWGQGTSVTVSSGGGGSGGGGSGGGGSDIVMTQAASSLSAS
LGDRVTISCRASQDISKYLNWYQQKPDGTVKLLIYYTSRLHSGVPSRFSG
SGSGTDYSLTIRNLEQEDIATYFCQQVYTLPWTFGGGTKLEIK or (SEQ ID NO: 2)
QVTLKESGPGILKPSQTLSLTCSFSGFSLNTSGMGVGWIRQPSGKGLEWL
AHIWWDDDKYYNPSLKSQLTISKDTSRNQVFLKITSVDTADSATYYCARR
ASHVSTVDSFDFWGQGTTLTVSSGGGGSGGGGSGGGGSDIQMTQTTSSLS
ASLGDRVTISCRASQDISNYLNWFQQKPDGTVKLLIYYTSRLHSGVPSKF
SGSGSGTDYSLTISNLEQEDIATYFCQQGYTLPWTFGGGTKLEIK.
[0047] A vector encoding the amino acid sequences described herein
may comprise a nucleic acid sequence that would encode AS, AT, or
both, such as ASAT (SEQ ID NO: 3) directly before the start codon.
These sequences are not translated as part of the CARs.
[0048] In an embodiment, the antigen binding domain comprises a
leader/signal sequence. The leader sequence may be positioned at
the amino terminus of the heavy chain variable region. The leader
sequence may comprise any suitable leader sequence. In embodiments
of the invention, the leader/signal sequence may comprise the
sequence as listed in Table 1 below. In the mature form of the T
cell, the leader sequence may not be present.
[0049] In an embodiment of the invention, the CAR comprises a
transmembrane domain. In an embodiment of the invention, the
transmembrane domain comprises CD8. The CD8 can comprise the
CD8.alpha. (CD8 alpha) hinge and transmembrane domain. In a
preferred embodiment, the CD8 is human. The CD8 may comprise less
than the whole CD8. In embodiments of the invention, the CD8 may
comprise the sequence as listed in Table 1 below.
[0050] In an embodiment of the invention, the CAR comprises an
intracellular T cell signaling domain comprising 4-1BB (CD137), CD3
zeta (0, or both. In a preferred embodiment, the CD3 zeta, 4-1BB,
or both is/are human. 4-1BB transmits a potent costimulatory signal
to T cells, promoting differentiation and enhancing long-term
survival of T lymphocytes. CD3.zeta. associates with TCRs to
produce a signal and contains immunoreceptor tyrosine-based
activation motifs (ITAMs). In an embodiment, the CAR lacks a 4-1BB
domain. In another embodiment, the CAR comprises a CD28 domain.
CD28 is a T cell marker important in T cell co-stimulation. The
4-1BB, CD28, CD3 zeta, or any of these may comprise less than the
whole 4-1BB or CD3 zeta, respectively. In embodiments of the
invention, the 4-1BB may comprise the sequence as listed in Table 1
below. In embodiments of the invention, the CD3 zeta may comprise
the sequence as listed in Table 1 below.
[0051] In an embodiment of the invention, the CAR comprises a
spacer. The spacer may be between any aforementioned domains. In an
embodiment, the CAR comprises an IgG heavy chain constant domain
(CH2CH3) spacer. In a further embodiment, the spacer can be between
the scFv and the transmembrane domain. In a preferred embodiment,
the sequence of the spacer, e.g., CH2CH3, is human. In embodiments
of the invention, the spacer may comprise the sequence as listed in
Table 1 below.
[0052] Embodiments of the invention comprise sequences as provided
in Table 1 below.
TABLE-US-00002 TABLE 1 SEQ ID Sequence NO: Segment Notes M 4 start
methionine ALPVTALLLPLALLLHAARP 5 signal peptide
QVTLKESGPGILKPSQTLSLTCS 6 scFv heavy chain FS GFSLX.sup.1TSGMG 7:
X.sup.1 as S scFv heavy chain: CDR1 8: X.sup.1 as N
VGWIRQPSGKGLEWLAH 9 scFv heavy chain IWWDDDK 10 scFv heavy chain:
CDR2 YYNPSLKSQLTISKDTSRNQVF 11: X.sup.2 as T scFv heavy chain
LKITSVDTADX.sup.2ATYYC 12: X.sup.2 as S
X.sup.3RRX.sup.4X.sup.5X.sup.6X.sup.7X.sup.8TX.sup.9DX.sup.10X.sup.11
13: X.sup.3 as S; scFv heavy chain: J region DX.sup.12 X.sup.4 as
P; X.sup.5 as R; (CDR3) X.sup.6 as no aa; X.sup.7 as no aa; X.sup.8
as G; X.sup.9 as M; X.sup.10 as A; X.sup.11 as M; X.sup.12 as Y 14:
X.sup.3 as A; X.sup.4 as A; X.sup.5 as S; X.sup.6 as H; X.sup.7 as
V; X.sup.8 as S; X.sup.9 as V; X.sup.10 as S; X.sup.11 as F;
X.sup.12 as F WGQGTX.sup.13X.sup.14TVSS 15: X.sup.13 as S; scFv
heavy chain X.sup.14 as V 16: X.sup.13 as T; X.sup.14 as L
GGGGSGGGGSGGGGS 17 scFv linker
DIX.sup.15MTQX.sup.16X.sup.17SSLSASLGDR 18: X.sup.15 as V; scFv
light chain VTISCRAS X.sup.16 as A; X.sup.17 as A 19: X.sup.15 as
Q; X.sup.16 as T; X.sup.17 as T QDISX.sup.18Y 20: X.sup.18 as K
scFv light chain: CDR1 21: X.sup.18 as N LNWX.sup.19QQKPDGTVKLLIY
22: X.sup.19 as Y scFv light chain 23: X.sup.19 as F YTS 24 scFv
light chain: CDR2 RLHSGVPSX.sup.20FSGSGSGTDYSL 25: X.sup.20 as R;
scFv light chain TIX.sup.21NLEQEDIATYFC X.sup.21 as R 26: X.sup.20
as K; X.sup.21 as S QQX.sup.22YTLPWT 27: X.sup.22 as V scFv light
chain: J region 28: X.sup.22 as G (CDR3) FGGGTKLEIK 29 scFv light
chain LEDP 30 spacer AEPKSPDKTHTCPPCPAPELLG 31 spacer CH2
GPSVFLFPPKPKDTLMISRTPEV TCVVVDVSHEDPEVKFNWYVD GVEVHNAKTKPREEQYNSTYR
VVSVLTVLHQDWLNGKEYKC KVSNKALPAPIEKTISKAK GQPREPQVYTLPPSRDELTKN 32
spacer CH3 QVSLTCLVKGFYPSDIAVEWES NGQPENNYKTTPPVLDSDGSFF
LYSKLTVDKSRWQQGNVFSCS VMHEALHNHYTQKSLSLSPGK KDPK 33 spacer SG 34
added amino acids due to vector design at BspEI site of vector
TTTPAPRPPTPAPTIASQPLSLRP 35 CD8 CD8.alpha. hinge
EACRPAAGGAVHTRGLDFACD IYIWAPLAGTCGVLLLSLVITLY 36 CD8 CD8
transmembrane domain C KRGRKKLLYIFKQPFMRPVQTT 37 4-1BB
intracellular domain QEEDGCSCRFPEEEEGGCEL RVKFSRSADAPAYKQGQNQLY 38
CD3.zeta. NELNLGRREEYDVLDKRRGRD PEMGGKPRRKNPQEGLYNELQ
KDKMAEAYSEIGMKGERRRGK GHDGLYQGLSTATKDTYDALH MQALPPR
[0053] Embodiments of the invention include the following sequences
in Table 2 that comprise the sequences presented in Table 1
above.
TABLE-US-00003 TABLE 2 Name Short 3G11 Long 3G11 Short 2D10 Long
2D10 SEQ ID NO: 39 40 41 42 Comprising 4 4 4 4 Table 1 5 5 5 5 SEQ
ID NOS: 6 6 6 6 7 7 8 8 9 9 9 9 10 10 10 10 11 11 12 12 13 13 14 14
15 15 16 16 17 17 17 17 18 18 19 19 20 20 21 21 22 22 23 23 24 24
24 24 25 25 26 26 27 27 28 28 29 29 29 29 30 30 31 31 32 32 33 33
34 34 34 34 35 35 35 35 36 36 36 36 37 37 37 37 38 38 38 38
[0054] Embodiments of the invention include the following sequences
in Table 3 that comprise the sequences presented in Table 1 above,
where the signal peptide is not present.
TABLE-US-00004 TABLE 3 Name Short 3G11 Long 3G11 Short 2D10 Long
2D10 SEQ ID NO: 43 44 45 46 Comprising 6 6 6 6 Table 1 7 7 8 8 SEQ
ID NOS: 9 9 9 9 10 10 10 10 11 11 12 12 13 13 14 14 15 15 16 16 17
17 17 17 18 18 19 19 20 20 21 21 22 22 23 23 24 24 24 24 25 25 26
26 27 27 28 28 29 29 29 29 30 30 31 31 32 32 33 33 34 34 34 34 35
35 35 35 36 36 36 36 37 37 37 37 38 38 38 38
[0055] Included in the scope of the invention are functional
portions of the inventive CARs described herein. The term
"functional portion" when used in reference to a CAR refers to any
part or fragment of the CAR of the invention, which part or
fragment retains the biological activity of the CAR of which it is
a part (the parent CAR). Functional portions encompass, for
example, those parts of a CAR that retain the ability to recognize
target cells, or detect, treat, or prevent a disease, to a similar
extent, the same extent, or to a higher extent, as the parent CAR.
In reference to the parent CAR, the functional portion can
comprise, for instance, about 10%, 25%, 30%, 50%, 68%, 80%, 90%,
95%, or more, of the parent CAR.
[0056] The functional portion can comprise additional amino acids
at the amino or carboxy terminus of the portion, or at both
termini, which additional amino acids are not found in the amino
acid sequence of the parent CAR. Desirably, the additional amino
acids do not interfere with the biological function of the
functional portion, e.g., recognize target cells, detect cancer,
treat or prevent cancer, etc. More desirably, the additional amino
acids enhance the biological activity, as compared to the
biological activity of the parent CAR.
[0057] Included in the scope of the invention are functional
variants of the inventive CARs described herein. The term
"functional variant" as used herein refers to a CAR, polypeptide,
or protein having substantial or significant sequence identity or
similarity to a parent CAR, which functional variant retains the
biological activity of the CAR of which it is a variant. Functional
variants encompass, for example, those variants of the CAR
described herein (the parent CAR) that retain the ability to
recognize target cells to a similar extent, the same extent, or to
a higher extent, as the parent CAR. In reference to the parent CAR,
the functional variant can, for instance, be at least about 30%,
50%, 75%, 80%, 90%, 98% or more identical in amino acid sequence to
the parent CAR.
[0058] A functional variant can, for example, comprise the amino
acid sequence of the parent CAR with at least one conservative
amino acid substitution. Alternatively or additionally, the
functional variants can comprise the amino acid sequence of the
parent CAR with at least one non-conservative amino acid
substitution. In this case, it is preferable for the
non-conservative amino acid substitution to not interfere with or
inhibit the biological activity of the functional variant. The
non-conservative amino acid substitution may enhance the biological
activity of the functional variant, such that the biological
activity of the functional variant is increased as compared to the
parent CAR.
[0059] Amino acid substitutions of the inventive CARs are
preferably conservative amino acid substitutions. Conservative
amino acid substitutions are known in the art, and include amino
acid substitutions in which one amino acid having certain physical
and/or chemical properties is exchanged for another amino acid that
has the same or similar chemical or physical properties. For
instance, the conservative amino acid substitution can be an
acidic/negatively charged polar amino acid substituted for another
acidic/negatively charged polar amino acid (e.g., Asp or Glu), an
amino acid with a nonpolar side chain substituted for another amino
acid with a nonpolar side chain (e.g., Ala, Gly, Val, Ile, Leu,
Met, Phe, Pro, Trp, Cys, Val, etc.), a basic/positively charged
polar amino acid substituted for another basic/positively charged
polar amino acid (e.g. Lys, His, Arg, etc.), an uncharged amino
acid with a polar side chain substituted for another uncharged
amino acid with a polar side chain (e.g., Asn, Gln, Ser, Thr, Tyr,
etc.), an amino acid with a beta-branched side-chain substituted
for another amino acid with a beta-branched side-chain (e.g., Ile,
Thr, and Val), an amino acid with an aromatic side-chain
substituted for another amino acid with an aromatic side chain
(e.g., His, Phe, Trp, and Tyr), etc.
[0060] Also, amino acids may be added or removed from the sequence
based on vector design. For example, SEQ ID NO: 34, added amino
acids due to vector design at BspEI site of vector, may be removed
from the CARs as described herein, e.g., removed from the sequences
in Table 2, Table 3, or both.
[0061] The CAR can consist essentially of the specified amino acid
sequence or sequences described herein, such that other components,
e.g., other amino acids, do not materially change the biological
activity of the functional variant.
[0062] The CARs of embodiments of the invention (including
functional portions and functional variants) can be of any length,
i.e., can comprise any number of amino acids, provided that the
CARs (or functional portions or functional variants thereof) retain
their biological activity, e.g., the ability to specifically bind
to antigen, detect diseased cells in a mammal, or treat or prevent
disease in a mammal, etc. For example, the CAR can be about 50 to
about 5000 amino acids long, such as 50, 70, 75, 100, 125, 150,
175, 200, 300, 400, 500, 600, 700, 800, 900, 1000 or more amino
acids in length.
[0063] The CARs of embodiments of the invention (including
functional portions and functional variants of the invention) can
comprise synthetic amino acids in place of one or more
naturally-occurring amino acids. Such synthetic amino acids are
known in the art, and include, for example, aminocyclohexane
carboxylic acid, norleucine, .alpha.-amino n-decanoic acid,
homoserine, S-acetylaminomethyl-cysteine, trans-3- and
trans-4-hydroxyproline, 4-aminophenylalanine, 4-nitrophenylalanine,
4-chlorophenylalanine, 4-carboxyphenylalanine, .beta.-phenylserine
.beta.-hydroxyphenylalanine, phenylglycine,
.alpha.-naphthylalanine, cyclohexylalanine, cyclohexylglycine,
indoline-2-carboxylic acid,
1,2,3,4-tetrahydroisoquinoline-3-carboxylic acid, aminomalonic
acid, aminomalonic acid monoamide, N'-benzyl-N'-methyl-lysine,
N',N'-dibenzyl-lysine, 6-hydroxylysine, ornithine,
.alpha.-aminocyclopentane carboxylic acid, .alpha.-aminocyclohexane
carboxylic acid, .alpha.-aminocycloheptane carboxylic acid,
.alpha.-(2-amino-2-norbornane)-carboxylic acid,
.alpha.,.gamma.-diaminobutyric acid,
.alpha.,.beta.-diaminopropionic acid, homophenylalanine, and
.alpha.-tert-butylglycine.
[0064] The CARs of embodiments of the invention (including
functional portions and functional variants) can be glycosylated,
amidated, carboxylated, phosphorylated, esterified, N-acylated,
cyclized via, e.g., a disulfide bridge, or converted into an acid
addition salt and/or optionally dimerized or polymerized, or
conjugated.
[0065] The CARs of embodiments of the invention (including
functional portions and functional variants thereof) can be
obtained by methods known in the art. The CARs may be made by any
suitable method of making polypeptides or proteins. Suitable
methods of de novo synthesizing polypeptides and proteins are
described in references, such as Chan et al., Fmoc Solid Phase
Peptide Synthesis, Oxford University Press, Oxford, United Kingdom,
2000; Peptide and Protein Drug Analysis, ed. Reid, R., Marcel
Dekker, Inc., 2000; Epitope Mapping, ed. Westwood et al., Oxford
University Press, Oxford, United Kingdom, 2001; and U.S. Pat. No.
5,449,752. Also, polypeptides and proteins can be recombinantly
produced using the nucleic acids described herein using standard
recombinant methods. See, for instance, Sambrook et al., Molecular
Cloning: A Laboratory Manual, 3.sup.rd ed., Cold Spring Harbor
Press, Cold Spring Harbor, N.Y. 2001; and Ausubel et al., Current
Protocols in Molecular Biology, Greene Publishing Associates and
John Wiley & Sons, N Y, 1994. Further, some of the CARs of the
invention (including functional portions and functional variants
thereof) can be isolated and/or purified from a source, such as a
plant, a bacterium, an insect, a mammal, e.g., a rat, a human, etc.
Methods of isolation and purification are well-known in the art.
Alternatively, the CARs described herein (including functional
portions and functional variants thereof) can be commercially
synthesized by companies. In this respect, the inventive CARs can
be synthetic, recombinant, isolated, and/or purified.
[0066] An embodiment of the invention further provides an antibody,
or antigen binding portion thereof, which specifically binds to an
epitope of the CARs of the invention. The antibody can be any type
of immunoglobulin that is known in the art. For instance, the
antibody can be of any isotype, e.g., IgA, IgD, IgE, IgG, IgM, etc.
The antibody can be monoclonal or polyclonal. The antibody can be a
naturally-occurring antibody, e.g., an antibody isolated and/or
purified from a mammal, e.g., mouse, rabbit, goat, horse, chicken,
hamster, human, etc. Alternatively, the antibody can be a
genetically-engineered antibody, e.g., a humanized antibody or a
chimeric antibody. The antibody can be in monomeric or polymeric
form. Also, the antibody can have any level of affinity or avidity
for the functional portion of the inventive CAR.
[0067] Methods of testing antibodies for the ability to bind to any
functional portion of the inventive CAR are known in the art and
include any antibody-antigen binding assay, such as, for example,
radioimmunoassay (RIA), ELISA, Western blot, immunoprecipitation,
and competitive inhibition assays (see, e.g., Janeway et al.,
infra, U.S. Patent Application Publication No. 2002/0197266 A1, and
U.S. Pat. No. 7,338,929).
[0068] Suitable methods of making antibodies are known in the art.
For instance, standard hybridoma methods are described in, e.g.,
Kohler and Milstein, Eur. J. Immunol., 5, 511-519 (1976), Harlow
and Lane (eds.), Antibodies: A Laboratory Manual, CSH Press (1988),
and C. A. Janeway et al. (eds.), Immunobiology, 5.sup.th Ed.,
Garland Publishing, New York, N.Y. (2001)). Alternatively, other
methods, such as EBV-hybridoma methods (Haskard and Archer, J.
Immunol. Methods, 74(2), 361-67 (1984), and Roder et al., Methods
Enzymol., 121, 140-67 (1986)), and bacteriophage vector expression
systems (see, e.g., Huse et al., Science, 246, 1275-81 (1989)) are
known in the art. Further, methods of producing antibodies in
non-human animals are described in, e.g., U.S. Pat. Nos. 5,545,806,
5,569,825, and 5,714,352, U.S. Patent Application Publication No.
2002/0197266 A1, and U.S. Pat. No. 7,338,929).
[0069] Phage display furthermore can be used to generate an
antibody. In this regard, phage libraries encoding antigen-binding
variable (V) domains of antibodies can be generated using standard
molecular biology and recombinant DNA techniques (see, e.g.,
Sambrook et al., supra, and Ausubel et al., supra). Phage encoding
a variable region with the desired specificity are selected for
specific binding to the desired antigen, and a complete or partial
antibody is reconstituted comprising the selected variable domain.
Nucleic acid sequences encoding the reconstituted antibody are
introduced into a suitable cell line, such as a myeloma cell used
for hybridoma production, such that antibodies having the
characteristics of monoclonal antibodies are secreted by the cell
(see, e.g., Janeway et al., supra, Huse et al., supra, and U.S.
Pat. No. 6,265,150).
[0070] Antibodies can be produced by transgenic mice that are
transgenic for specific heavy and light chain immunoglobulin genes.
Such methods are known in the art and described in, for example
U.S. Pat. Nos. 5,545,806 and 5,569,825, and Janeway et al.,
supra.
[0071] Methods for generating humanized antibodies are well known
in the art and are described in detail in, for example, Janeway et
al., supra, U.S. Pat. Nos. 5,225,539, 5,585,089 and 5,693,761,
European Patent No. 0239400 B1, and United Kingdom Patent No.
2188638. Humanized antibodies can also be generated using the
antibody resurfacing technology described in U.S. Pat. No.
5,639,641 and Pedersen et al., J. Mol. Biol., 235, 959-973
(1994).
[0072] An embodiment of the invention also provides antigen binding
portions of any of the antibodies described herein. The antigen
binding portion can be any portion that has at least one antigen
binding site, such as Fab, F(ab').sub.2, dsFv, sFv, diabodies, and
triabodies.
[0073] A single-chain variable region fragment (sFv) antibody
fragment can be generated using routine recombinant DNA technology
techniques (see, e.g., Janeway et al., supra). Similarly,
disulfide-stabilized variable region fragments (dsFv) can be
prepared by recombinant DNA technology (see, e.g., Reiter et al.,
Protein Engineering, 7, 697-704 (1994)). Antibody fragments of the
invention, however, are not limited to these exemplary types of
antibody fragments.
[0074] Also, the antibody, or antigen binding portion thereof, can
be modified to comprise a detectable label, such as, for instance,
a radioisotope, a fluorophore (e.g., fluorescein isothiocyanate
(FITC), phycoerythrin (PE)), an enzyme (e.g., alkaline phosphatase,
horseradish peroxidase), and element particles (e.g., gold
particles).
[0075] Further provided by an embodiment of the invention is a
nucleic acid comprising a nucleotide sequence encoding any of the
CARs described herein (including functional portions and functional
variants thereof). The nucleic acids of the invention may comprise
a nucleotide sequence encoding any of the leader sequences, antigen
binding domains, transmembrane domains, and/or intracellular T cell
signaling domains described herein.
[0076] In some embodiments, the nucleotide sequence may be
codon-optimized. Without being bound to a particular theory, it is
believed that codon optimization of the nucleotide sequence
increases the translation efficiency of the mRNA transcripts. Codon
optimization of the nucleotide sequence may involve substituting a
native codon for another codon that encodes the same amino acid,
but can be translated by tRNA that is more readily available within
a cell, thus increasing translation efficiency. Optimization of the
nucleotide sequence may also reduce secondary mRNA structures that
would interfere with translation, thus increasing translation
efficiency.
[0077] In an embodiment of the invention, the nucleic acid may
comprise a codon-optimized nucleotide sequence that encodes the
antigen binding domain of the inventive CAR. In another embodiment
of the invention, the nucleic acid may comprise a codon-optimized
nucleotide sequence that encodes any of the CARs described herein
(including functional portions and functional variants
thereof).
[0078] "Nucleic acid" as used herein includes "polynucleotide,"
"oligonucleotide," and "nucleic acid molecule," and generally means
a polymer of DNA or RNA, which can be single-stranded or
double-stranded, synthesized or obtained (e.g., isolated and/or
purified) from natural sources, which can contain natural,
non-natural or altered nucleotides, and which can contain a
natural, non-natural or altered internucleotide linkage, such as a
phosphoroamidate linkage or a phosphorothioate linkage, instead of
the phosphodiester found between the nucleotides of an unmodified
oligonucleotide. In some embodiments, the nucleic acid does not
comprise any insertions, deletions, inversions, and/or
substitutions. However, it may be suitable in some instances, as
discussed herein, for the nucleic acid to comprise one or more
insertions, deletions, inversions, and/or substitutions.
[0079] The nucleic acids of an embodiment of the invention may be
recombinant. As used herein, the term "recombinant" refers to (i)
molecules that are constructed outside living cells by joining
natural or synthetic nucleic acid segments to nucleic acid
molecules that can replicate in a living cell, or (ii) molecules
that result from the replication of those described in (i) above.
For purposes herein, the replication can be in vitro replication or
in vivo replication.
[0080] A recombinant nucleic acid may be one that has a sequence
that is not naturally occurring or has a sequence that is made by
an artificial combination of two otherwise separated segments of
sequence. This artificial combination is often accomplished by
chemical synthesis or, more commonly, by the artificial
manipulation of isolated segments of nucleic acids, e.g., by
genetic engineering techniques, such as those described in Sambrook
et al., supra. The nucleic acids can be constructed based on
chemical synthesis and/or enzymatic ligation reactions using
procedures known in the art. See, for example, Sambrook et al.,
supra, and Ausubel et al., supra. For example, a nucleic acid can
be chemically synthesized using naturally occurring nucleotides or
variously modified nucleotides designed to increase the biological
stability of the molecules or to increase the physical stability of
the duplex formed upon hybridization (e.g., phosphorothioate
derivatives and acridine substituted nucleotides). Examples of
modified nucleotides that can be used to generate the nucleic acids
include, but are not limited to, 5-fluorouracil, 5-bromouracil,
5-chlorouracil, 5-iodouracil, hypoxanthine, xanthine,
4-acetylcytosine, 5-(carboxyhydroxymethyl) uracil,
5-carboxymethylaminomethyl-2-thiouridine,
5-carboxymethylaminomethyluracil, dihydrouracil,
beta-D-galactosylqueosine, inosine, N.sup.6-isopentenyladenine,
1-methylguanine, 1-methylinosine, 2,2-dimethylguanine,
2-methyladenine, 2-methylguanine, 3-methylcytosine,
5-methylcytosine, N.sup.6-substituted adenine, 7-methylguanine,
5-methylaminomethyluracil, 5-methoxyaminomethyl-2-thiouracil,
beta-D-mannosylqueosine, 5'-methoxycarboxymethyluracil,
5-methoxyuracil, 2-methylthio-N.sup.6-isopentenyladenine,
uracil-5-oxyacetic acid (v), wybutoxosine, pseudouracil, queosine,
2-thiocytosine, 5-methyl-2-thiouracil, 2-thiouracil, 4-thiouracil,
5-methyluracil, uracil-5-oxyacetic acid methylester,
3-(3-amino-3-N-2-carboxypropyl) uracil, and 2,6-diaminopurine.
Alternatively, one or more of the nucleic acids of the invention
can be purchased from companies, such as Integrated DNA
Technologies (Coralville, Iowa, USA).
[0081] The nucleic acid can comprise any isolated or purified
nucleotide sequence which encodes any of the CARs or functional
portions or functional variants thereof. Alternatively, the
nucleotide sequence can comprise a nucleotide sequence which is
degenerate to any of the sequences or a combination of degenerate
sequences.
[0082] An embodiment of the invention also provides an isolated or
purified nucleic acid comprising a nucleotide sequence which is
complementary to the nucleotide sequence of any of the nucleic
acids described herein or a nucleotide sequence which hybridizes
under stringent conditions to the nucleotide sequence of any of the
nucleic acids described herein.
[0083] The nucleotide sequence which hybridizes under stringent
conditions may hybridize under high stringency conditions. By "high
stringency conditions" is meant that the nucleotide sequence
specifically hybridizes to a target sequence (the nucleotide
sequence of any of the nucleic acids described herein) in an amount
that is detectably stronger than non-specific hybridization. High
stringency conditions include conditions which would distinguish a
polynucleotide with an exact complementary sequence, or one
containing only a few scattered mismatches from a random sequence
that happened to have a few small regions (e.g., 3-10 bases) that
matched the nucleotide sequence. Such small regions of
complementarity are more easily melted than a full-length
complement of 14-17 or more bases, and high stringency
hybridization makes them easily distinguishable. Relatively high
stringency conditions would include, for example, low salt and/or
high temperature conditions, such as provided by about 0.02-0.1 M
NaCl or the equivalent, at temperatures of about 50-70.degree. C.
Such high stringency conditions tolerate little, if any, mismatch
between the nucleotide sequence and the template or target strand,
and are particularly suitable for detecting expression of any of
the inventive CARs. It is generally appreciated that conditions can
be rendered more stringent by the addition of increasing amounts of
formamide.
[0084] The invention also provides a nucleic acid comprising a
nucleotide sequence that is at least about 70% or more, e.g., about
80%, about 90%, about 91%, about 92%, about 93%, about 94%, about
95%, about 96%, about 97%, about 98%, or about 99% identical to any
of the nucleic acids described herein.
[0085] In an embodiment, the nucleic acids of the invention can be
incorporated into a recombinant expression vector. In this regard,
an embodiment of the invention provides recombinant expression
vectors comprising any of the nucleic acids of the invention. For
purposes herein, the term "recombinant expression vector" means a
genetically-modified oligonucleotide or polynucleotide construct
that permits the expression of an mRNA, protein, polypeptide, or
peptide by a host cell, when the construct comprises a nucleotide
sequence encoding the mRNA, protein, polypeptide, or peptide, and
the vector is contacted with the cell under conditions sufficient
to have the mRNA, protein, polypeptide, or peptide expressed within
the cell. The vectors of the invention are not naturally-occurring
as a whole. However, parts of the vectors can be
naturally-occurring. The inventive recombinant expression vectors
can comprise any type of nucleotides, including, but not limited to
DNA and RNA, which can be single-stranded or double-stranded,
synthesized or obtained in part from natural sources, and which can
contain natural, non-natural or altered nucleotides. The
recombinant expression vectors can comprise naturally-occurring or
non-naturally-occurring internucleotide linkages, or both types of
linkages. Preferably, the non-naturally occurring or altered
nucleotides or internucleotide linkages do not hinder the
transcription or replication of the vector.
[0086] In an embodiment, the recombinant expression vector of the
invention can be any suitable recombinant expression vector, and
can be used to transform or transfect any suitable host cell.
Suitable vectors include those designed for propagation and
expansion or for expression or both, such as plasmids and viruses.
The vector can be selected from the group consisting of the pUC
series (Fermentas Life Sciences, Glen Burnie, Md.), the pBluescript
series (Stratagene, LaJolla, Calif.), the pET series (Novagen,
Madison, Wis.), the pGEX series (Pharmacia Biotech, Uppsala,
Sweden), and the pEX series (Clontech, Palo Alto, Calif.).
Bacteriophage vectors, such as kGT10, 2 GT11, kZapII (Stratagene),
2 EMBL4, and kNM1149, also can be used. Examples of plant
expression vectors include pBI01, pBI101.2, pBI101.3, pBI121 and
pBIN19 (Clontech). Examples of animal expression vectors include
pEUK-Cl, pMAM, and pMAMneo (Clontech). The recombinant expression
vector may be a viral vector, e.g., a retroviral vector or a
lentiviral vector.
[0087] A number of transfection techniques are generally known in
the art (see, e.g., Graham et al., Virology, 52: 456-467 (1973);
Sambrook et al., supra; Davis et al., Basic Methods in Molecular
Biology, Elsevier (1986); and Chu et al., Gene, 13: 97 (1981).
Transfection methods include calcium phosphate co-precipitation
(see, e.g., Graham et al., supra), direct micro injection into
cultured cells (see, e.g., Capecchi, Cell, 22: 479-488 (1980)),
electroporation (see, e.g., Shigekawa et al., BioTechniques, 6:
742-751 (1988)), liposome mediated gene transfer (see, e.g.,
Mannino et al., BioTechniques, 6: 682-690 (1988)), lipid mediated
transduction (see, e.g., Feigner et al., Proc. Natl. Acad. Sci.
USA, 84: 7413-7417 (1987)), and nucleic acid delivery using high
velocity microprojectiles (see, e.g., Klein et al., Nature, 327:
70-73 (1987)).
[0088] In an embodiment, the recombinant expression vectors of the
invention can be prepared using standard recombinant DNA techniques
described in, for example, Sambrook et al., supra, and Ausubel et
al., supra. Constructs of expression vectors, which are circular or
linear, can be prepared to contain a replication system functional
in a prokaryotic or eukaryotic host cell. Replication systems can
be derived, e.g., from ColEl, 2.mu. plasmid, .lamda., SV40, bovine
papilloma virus, and the like.
[0089] The recombinant expression vector may comprise regulatory
sequences, such as transcription and translation initiation and
termination codons, which are specific to the type of host cell
(e.g., bacterium, fungus, plant, or animal) into which the vector
is to be introduced, as appropriate, and taking into consideration
whether the vector is DNA- or RNA-based. The recombinant expression
vector may comprise restriction sites to facilitate cloning.
[0090] The recombinant expression vector can include one or more
marker genes, which allow for selection of transformed or
transfected host cells. Marker genes include biocide resistance,
e.g., resistance to antibiotics, heavy metals, etc.,
complementation in an auxotrophic host to provide prototrophy, and
the like. Suitable marker genes for the inventive expression
vectors include, for instance, neomycin/G418 resistance genes,
hygromycin resistance genes, histidinol resistance genes,
tetracycline resistance genes, and ampicillin resistance genes.
[0091] The recombinant expression vector can comprise a native or
nonnative promoter operably linked to the nucleotide sequence
encoding the CAR (including functional portions and functional
variants thereof), or to the nucleotide sequence which is
complementary to or which hybridizes to the nucleotide sequence
encoding the CAR. The selection of promoters, e.g., strong, weak,
inducible, tissue-specific and developmental-specific, is within
the ordinary skill of the artisan. Similarly, the combining of a
nucleotide sequence with a promoter is also within the skill of the
artisan. The promoter can be a non-viral promoter or a viral
promoter, e.g., a cytomegalovirus (CMV) promoter, an SV40 promoter,
an RSV promoter, or a promoter found in the long-terminal repeat of
the murine stem cell virus.
[0092] The inventive recombinant expression vectors can be designed
for either transient expression, for stable expression, or for
both. Also, the recombinant expression vectors can be made for
constitutive expression or for inducible expression.
[0093] Further, the recombinant expression vectors can be made to
include a suicide gene. As used herein, the term "suicide gene"
refers to a gene that causes the cell expressing the suicide gene
to die. The suicide gene can be a gene that confers sensitivity to
an agent, e.g., a drug, upon the cell in which the gene is
expressed, and causes the cell to die when the cell is contacted
with or exposed to the agent. Suicide genes are known in the art
(see, for example, Suicide Gene Therapy: Methods and Reviews,
Springer, Caroline J. (Cancer Research UK Centre for Cancer
Therapeutics at the Institute of Cancer Research, Sutton, Surrey,
UK), Humana Press, 2004) and include, for example, the Herpes
Simplex Virus (HSV) thymidine kinase (TK) gene, cytosine daminase,
purine nucleoside phosphorylase, and nitroreductase.
[0094] Included in the scope of the invention are conjugates, e.g.,
bioconjugates, comprising any of the inventive CARs (including any
of the functional portions or variants thereof), nucleic acids,
recombinant expression vectors, host cells, populations of host
cells, or antibodies, or antigen binding portions thereof.
Conjugates, as well as methods of synthesizing conjugates in
general, are known in the art (See, for instance, Hudecz, F.,
Methods Mol. Biol. 298: 209-223 (2005) and Kirin et al., Inorg
Chem. 44(15): 5405-5415 (2005)).
[0095] An embodiment of the invention further provides a host cell
comprising any of the recombinant expression vectors described
herein. As used herein, the term "host cell" refers to any type of
cell that can contain the inventive recombinant expression vector.
The host cell can be a eukaryotic cell, e.g., plant, animal, fungi,
or algae, or can be a prokaryotic cell, e.g., bacteria or protozoa.
The host cell can be a cultured cell or a primary cell, i.e.,
isolated directly from an organism, e.g., a human. The host cell
can be an adherent cell or a suspended cell, i.e., a cell that
grows in suspension. Suitable host cells are known in the art and
include, for instance, DH5.alpha. E. coli cells, Chinese hamster
ovarian cells, monkey VERO cells, COS cells, HEK293 cells, and the
like. For purposes of amplifying or replicating the recombinant
expression vector, the host cell may be a prokaryotic cell, e.g., a
DH5.alpha. cell. For purposes of producing a recombinant CAR, the
host cell may be a mammalian cell. The host cell may be a human
cell. While the host cell can be of any cell type, can originate
from any type of tissue, and can be of any developmental stage, the
host cell may be a peripheral blood lymphocyte (PBL) or a
peripheral blood mononuclear cell (PBMC). The host cell may be a T
cell.
[0096] For purposes herein, the T cell can be any T cell, such as a
cultured T cell, e.g., a primary T cell, or a T cell from a
cultured T cell line, e.g., Jurkat, SupTi, etc., or a T cell
obtained from a mammal. If obtained from a mammal, the T cell can
be obtained from numerous sources, including but not limited to
blood, bone marrow, lymph node, the thymus, or other tissues or
fluids. T cells can also be enriched for or purified. The T cell
may be a human T cell. The T cell may be a T cell isolated from a
human. The T cell can be any type of T cell and can be of any
developmental stage, including but not limited to,
CD4.sup.+/CD8.sup.+ double positive T cells, CD4.sup.+ helper T
cells, e.g., Th.sub.1 and Th.sub.2 cells, CD8.sup.+ T cells (e.g.,
cytotoxic T cells), tumor infiltrating cells, memory T cells, nave
T cells, and the like. The T cell may be a CD8.sup.+ T cell or a
CD4.sup.+ T cell.
[0097] In an embodiment, the CARs as described herein can be used
in suitable non-T cells. Such cells are those with an
immune-effector function, such as, for example, NK cells, and
T-like cells generated from pluripotent stem cells.
[0098] Also provided by an embodiment of the invention is a
population of cells comprising at least one host cell described
herein. The population of cells can be a heterogeneous population
comprising the host cell comprising any of the recombinant
expression vectors described, in addition to at least one other
cell, e.g., a host cell (e.g., a T cell), which does not comprise
any of the recombinant expression vectors, or a cell other than a T
cell, e.g., a B cell, a macrophage, a neutrophil, an erythrocyte, a
hepatocyte, an endothelial cell, an epithelial cell, a muscle cell,
a brain cell, etc. Alternatively, the population of cells can be a
substantially homogeneous population, in which the population
comprises mainly host cells (e.g., consisting essentially of)
comprising the recombinant expression vector. The population also
can be a clonal population of cells, in which all cells of the
population are clones of a single host cell comprising a
recombinant expression vector, such that all cells of the
population comprise the recombinant expression vector. In one
embodiment of the invention, the population of cells is a clonal
population comprising host cells comprising a recombinant
expression vector as described herein.
[0099] CARs (including functional portions and variants thereof),
nucleic acids, recombinant expression vectors, host cells
(including populations thereof), and antibodies (including antigen
binding portions thereof), all of which are collectively referred
to as "inventive CAR materials" hereinafter, can be isolated and/or
purified. The term "isolated" as used herein means having been
removed from its natural environment. The term "purified" or
"isolated" does not require absolute purity or isolation; rather,
it is intended as a relative term. Thus, for example, a purified
(or isolated) host cell preparation is one in which the host cell
is more pure than cells in their natural environment within the
body. Such host cells may be produced, for example, by standard
purification techniques. In some embodiments, a preparation of a
host cell is purified such that the host cell represents at least
about 50%, for example at least about 70%, of the total cell
content of the preparation. For example, the purity can be at least
about 50%, can be greater than about 60%, about 70% or about 80%,
or can be about 100%.
[0100] The inventive CAR materials can be formulated into a
composition, such as a pharmaceutical composition. In this regard,
an embodiment of the invention provides a pharmaceutical
composition comprising any of the CARs, functional portions,
functional variants, nucleic acids, expression vectors, host cells
(including populations thereof), and antibodies (including antigen
binding portions thereof), and a pharmaceutically acceptable
carrier. The inventive pharmaceutical compositions containing any
of the inventive CAR materials can comprise more than one inventive
CAR material, e.g., a CAR and a nucleic acid, or two or more
different CARs. Alternatively, the pharmaceutical composition can
comprise an inventive CAR material in combination with other
pharmaceutically active agents or drugs, such as chemotherapeutic
agents, e.g., asparaginase, busulfan, carboplatin, cisplatin,
daunorubicin, doxorubicin, fluorouracil, gemcitabine, hydroxyurea,
methotrexate, paclitaxel, rituximab, vinblastine, vincristine, etc.
In a preferred embodiment, the pharmaceutical composition comprises
the inventive host cell or populations thereof.
[0101] The inventive CAR materials can be provided in the form of a
salt, e.g., a pharmaceutically acceptable salt. Suitable
pharmaceutically acceptable acid addition salts include those
derived from mineral acids, such as hydrochloric, hydrobromic,
phosphoric, metaphosphoric, nitric, and sulphuric acids, and
organic acids, such as tartaric, acetic, citric, malic, lactic,
fumaric, benzoic, glycolic, gluconic, succinic, and arylsulphonic
acids, for example, p-toluenesulphonic acid.
[0102] With respect to pharmaceutical compositions, the
pharmaceutically acceptable carrier can be any of those
conventionally used and is limited only by chemico-physical
considerations, such as solubility and lack of reactivity with the
active agent(s), and by the route of administration. The
pharmaceutically acceptable carriers described herein, for example,
vehicles, adjuvants, excipients, and diluents, are well-known to
those skilled in the art and are readily available to the public.
It is preferred that the pharmaceutically acceptable carrier be one
which is chemically inert to the active agent(s) and one which has
no detrimental side effects or toxicity under the conditions of
use.
[0103] The choice of carrier will be determined in part by the
particular inventive CAR material, as well as by the particular
method used to administer the inventive CAR material. Accordingly,
there are a variety of suitable formulations of the pharmaceutical
composition of the invention. Preservatives may be used. Suitable
preservatives may include, for example, methylparaben,
propylparaben, sodium benzoate, and benzalkonium chloride. A
mixture of two or more preservatives optionally may be used. The
preservative or mixtures thereof are typically present in an amount
of about 0.0001% to about 2% by weight of the total
composition.
[0104] Suitable buffering agents may include, for example, citric
acid, sodium citrate, phosphoric acid, potassium phosphate, and
various other acids and salts. A mixture of two or more buffering
agents optionally may be used. The buffering agent or mixtures
thereof are typically present in an amount of about 0.001% to about
4% by weight of the total composition.
[0105] The concentration of inventive CAR material in the
pharmaceutical formulations can vary, e.g., from less than about
1%, usually at or at least about 10%, to as much as about 20% to
about 50% or more by weight, and can be selected primarily by fluid
volumes, and viscosities, in accordance with the particular mode of
administration selected.
[0106] Methods for preparing administrable (e.g., parenterally
administrable) compositions are known or apparent to those skilled
in the art and are described in more detail in, for example,
Remington: The Science and Practice of Pharmacy, Lippincott
Williams & Wilkins; 21st ed. (May 1, 2005).
[0107] The following formulations for oral, aerosol, parenteral
(e.g., subcutaneous, intravenous, intraarterial, intramuscular,
intradermal, interperitoneal, and intrathecal), and topical
administration are merely exemplary and are in no way limiting.
More than one route can be used to administer the inventive CAR
materials, and in certain instances, a particular route can provide
a more immediate and more effective response than another
route.
[0108] Formulations suitable for oral administration can comprise
or consist of (a) liquid solutions, such as an effective amount of
the inventive CAR material dissolved in diluents, such as water,
saline, or orange juice; (b) capsules, sachets, tablets, lozenges,
and troches, each containing a predetermined amount of the active
ingredient, as solids or granules; (c) powders; (d) suspensions in
an appropriate liquid; and (e) suitable emulsions. Liquid
formulations may include diluents, such as water and alcohols, for
example, ethanol, benzyl alcohol, and the polyethylene alcohols,
either with or without the addition of a pharmaceutically
acceptable surfactant. Capsule forms can be of the ordinary hard or
softshelled gelatin type containing, for example, surfactants,
lubricants, and inert fillers, such as lactose, sucrose, calcium
phosphate, and corn starch. Tablet forms can include one or more of
lactose, sucrose, mannitol, corn starch, potato starch, alginic
acid, microcrystalline cellulose, acacia, gelatin, guar gum,
colloidal silicon dioxide, croscarmellose sodium, talc, magnesium
stearate, calcium stearate, zinc stearate, stearic acid, and other
excipients, colorants, diluents, buffering agents, disintegrating
agents, moistening agents, preservatives, flavoring agents, and
other pharmacologically compatible excipients. Lozenge forms can
comprise the inventive CAR material in a flavor, usually sucrose
and acacia or tragacanth, as well as pastilles comprising the
inventive CAR material in an inert base, such as gelatin and
glycerin, or sucrose and acacia, emulsions, gels, and the like
containing, in addition to, such excipients as are known in the
art.
[0109] Formulations suitable for parenteral administration include
aqueous and nonaqueous isotonic sterile injection solutions, which
can contain antioxidants, buffers, bacteriostats, and solutes that
render the formulation isotonic with the blood of the intended
recipient, and aqueous and nonaqueous sterile suspensions that can
include suspending agents, solubilizers, thickening agents,
stabilizers, and preservatives. The inventive CAR material can be
administered in a physiologically acceptable diluent in a
pharmaceutical carrier, such as a sterile liquid or mixture of
liquids, including water, saline, aqueous dextrose and related
sugar solutions, an alcohol, such as ethanol or hexadecyl alcohol,
a glycol, such as propylene glycol or polyethylene glycol,
dimethylsulfoxide, glycerol, ketals such as
2,2-dimethyl-1,3-dioxolane-4-methanol, ethers, poly(ethyleneglycol)
400, oils, fatty acids, fatty acid esters or glycerides, or
acetylated fatty acid glycerides with or without the addition of a
pharmaceutically acceptable surfactant, such as a soap or a
detergent, suspending agent, such as pectin, carbomers,
methylcellulose, hydroxypropylmethylcellulose, or
carboxymethylcellulose, or emulsifying agents and other
pharmaceutical adjuvants.
[0110] Oils, which can be used in parenteral formulations, include
petroleum, animal, vegetable, or synthetic oils. Specific examples
of oils include peanut, soybean, sesame, cottonseed, corn, olive,
petrolatum, and mineral. Suitable fatty acids for use in parenteral
formulations include oleic acid, stearic acid, and isostearic acid.
Ethyl oleate and isopropyl myristate are examples of suitable fatty
acid esters.
[0111] Suitable soaps for use in parenteral formulations include
fatty alkali metal, ammonium, and triethanolamine salts, and
suitable detergents include (a) cationic detergents such as, for
example, dimethyl dialkyl ammonium halides, and alkyl pyridinium
halides, (b) anionic detergents such as, for example, alkyl, aryl,
and olefin sulfonates, alkyl, olefin, ether, and monoglyceride
sulfates, and sulfosuccinates, (c) nonionic detergents such as, for
example, fatty amine oxides, fatty acid alkanolamides, and
polyoxyethylenepolypropylene copolymers, (d) amphoteric detergents
such as, for example, alkyl-.beta.-aminopropionates, and
2-alkyl-imidazoline quaternary ammonium salts, and (e) mixtures
thereof.
[0112] The parenteral formulations will typically contain, for
example, from about 0.5% to about 25% by weight of the inventive
CAR material in solution. Preservatives and buffers may be used. In
order to minimize or eliminate irritation at the site of injection,
such compositions may contain one or more nonionic surfactants
having, for example, a hydrophile-lipophile balance (HLB) of from
about 12 to about 17. The quantity of surfactant in such
formulations will typically range, for example, from about 5% to
about 15% by weight. Suitable surfactants include polyethylene
glycol sorbitan fatty acid esters, such as sorbitan monooleate and
the high molecular weight adducts of ethylene oxide with a
hydrophobic base, formed by the condensation of propylene oxide
with propylene glycol. The parenteral formulations can be presented
in unit-dose or multi-dose sealed containers, such as ampoules and
vials, and can be stored in a freeze-dried (lyophilized) condition
requiring only the addition of the sterile liquid excipient, for
example, water, for injections, immediately prior to use.
Extemporaneous injection solutions and suspensions can be prepared
from sterile powders, granules, and tablets of the kind previously
described.
[0113] Injectable formulations are in accordance with an embodiment
of the invention. The requirements for effective pharmaceutical
carriers for injectable compositions are well-known to those of
ordinary skill in the art (see, e.g., Pharmaceutics and Pharmacy
Practice, J.B. Lippincott Company, Philadelphia, Pa., Banker and
Chalmers, eds., pages 238-250 (1982), and ASHP Handbook on
Injectable Drugs, Toissel, 4th ed., pages 622-630 (1986)).
[0114] Topical formulations, including those that are useful for
transdermal drug release, are well known to those of skill in the
art and are suitable in the context of embodiments of the invention
for application to skin. The inventive CAR material, alone or in
combination with other suitable components, can be made into
aerosol formulations to be administered via inhalation. These
aerosol formulations can be placed into pressurized acceptable
propellants, such as dichlorodifluoromethane, propane, nitrogen,
and the like. They also may be formulated as pharmaceuticals for
non-pressured preparations, such as in a nebulizer or an atomizer.
Such spray formulations also may be used to spray mucosa.
[0115] An "effective amount" or "an amount effective to treat"
refers to a dose that is adequate to prevent or treat cancer in an
individual. Amounts effective for a therapeutic or prophylactic use
will depend on, for example, the stage and severity of the disease
or disorder being treated, the age, weight, and general state of
health of the patient, and the judgment of the prescribing
physician. The size of the dose will also be determined by the
active selected, method of administration, timing and frequency of
administration, the existence, nature, and extent of any adverse
side-effects that might accompany the administration of a
particular active, and the desired physiological effect. It will be
appreciated by one of skill in the art that various diseases or
disorders could require prolonged treatment involving multiple
administrations, perhaps using the inventive CAR materials in each
or various rounds of administration. By way of example and not
intending to limit the invention, the dose of the inventive CAR
material can be about 0.001 to about 1000 mg/kg body weight of the
subject being treated/day, from about 0.01 to about 10 mg/kg body
weight/day, about 0.01 mg to about 1 mg/kg body weight/day. In an
embodiment of the invention, the dose may be from about
1.times.10.sup.4 to about 1.times.10.sup.8 cells expressing the
inventive CAR material per kg body weight. When the inventive CAR
material is a host cell, an exemplary dose of host cells may be a
minimum of one million cells (1 mg cells/dose). When the inventive
CAR material is a nucleic acid packaged in a virus, an exemplary
dose of virus may be 1 ng/dose.
[0116] For purposes of the invention, the amount or dose of the
inventive CAR material administered should be sufficient to effect
a therapeutic or prophylactic response in the subject or animal
over a reasonable time frame. For example, the dose of the
inventive CAR material should be sufficient to bind to antigen, or
detect, treat or prevent disease in a period of from about 2 hours
or longer, e.g., about 12 to about 24 or more hours, from the time
of administration. In certain embodiments, the time period could be
even longer. The dose will be determined by the efficacy of the
particular inventive CAR material and the condition of the animal
(e.g., human), as well as the body weight of the animal (e.g.,
human) to be treated.
[0117] For purposes of the invention, an assay, which comprises,
for example, comparing the extent to which target cells are lysed
and/or IFN-.gamma. is secreted by T cells expressing the inventive
CAR upon administration of a given dose of such T cells to a
mammal, among a set of mammals of which is each given a different
dose of the T cells, could be used to determine a starting dose to
be administered to a mammal. The extent to which target cells are
lysed and/or IFN-.gamma. is secreted upon administration of a
certain dose can be assayed by methods known in the art.
[0118] In addition to the aforedescribed pharmaceutical
compositions, the inventive CAR materials can be formulated as
inclusion complexes, such as cyclodextrin inclusion complexes, or
liposomes. Liposomes can serve to target the inventive CAR
materials to a particular tissue. Liposomes also can be used to
increase the half-life of the inventive CAR materials. Many methods
are available for preparing liposomes, as described in, for
example, Szoka et al., Ann. Rev. Biophys. Bioeng., 9, 467 (1980)
and U.S. Pat. Nos. 4,235,871, 4,501,728, 4,837,028, and
5,019,369.
[0119] The delivery systems useful in the context of embodiments of
the invention may include time-released, delayed release, and
sustained release delivery systems such that the delivery of the
inventive composition occurs prior to, and with sufficient time to
cause, sensitization of the site to be treated. The inventive
composition can be used in conjunction with other therapeutic
agents or therapies. Such systems can avoid repeated
administrations of the inventive composition, thereby increasing
convenience to the subject and the physician, and may be
particularly suitable for certain composition embodiments of the
invention.
[0120] Many types of release delivery systems are available and
known to those of ordinary skill in the art. They include polymer
base systems such as poly(lactide-glycolide), copolyoxalates,
polycaprolactones, polyesteramides, polyorthoesters,
polyhydroxybutyric acid, and polyanhydrides. Microcapsules of the
foregoing polymers containing drugs are described in, for example,
U.S. Pat. No. 5,075,109. Delivery systems also include non-polymer
systems that are lipids including sterols such as cholesterol,
cholesterol esters, and fatty acids or neutral fats such as mono-
di- and tri-glycerides; hydrogel release systems; sylastic systems;
peptide based systems; wax coatings; compressed tablets using
conventional binders and excipients; partially fused implants; and
the like. Specific examples include, but are not limited to: (a)
erosional systems in which the active composition is contained in a
form within a matrix such as those described in U.S. Pat. Nos.
4,452,775, 4,667,014, 4,748,034, and 5,239,660 and (b) diffusional
systems in which an active component permeates at a controlled rate
from a polymer such as described in U.S. Pat. Nos. 3,832,253 and
3,854,480. In addition, pump-based hardware delivery systems can be
used, some of which are adapted for implantation.
[0121] One of ordinary skill in the art will readily appreciate
that the inventive CAR materials of the invention can be modified
in any number of ways, such that the therapeutic or prophylactic
efficacy of the inventive CAR materials is increased through the
modification. For instance, the inventive CAR materials can be
conjugated either directly or indirectly through a linking moiety
to a targeting moiety. The practice of conjugating compounds, e.g.,
inventive CAR materials, to targeting moieties is known in the art.
See, for instance, Wadwa et al., J. Drug Targeting 3: 111 (1995)
and U.S. Pat. No. 5,087,616.
[0122] Alternatively, the inventive CAR materials can be modified
into a depot form, such that the manner in which the inventive CAR
materials is released into the body to which it is administered is
controlled with respect to time and location within the body (see,
for example, U.S. Pat. No. 4,450,150). Depot forms of inventive CAR
materials can be, for example, an implantable composition
comprising the inventive CAR materials and a porous or non-porous
material, such as a polymer, wherein the inventive CAR materials
are encapsulated by or diffused throughout the material and/or
degradation of the non-porous material. The depot is then implanted
into the desired location within the body and the inventive CAR
materials are released from the implant at a predetermined
rate.
[0123] When the inventive CAR materials are administered with one
or more additional therapeutic agents, one or more additional
therapeutic agents can be coadministered to the mammal. By
"coadministering" is meant administering one or more additional
therapeutic agents and the inventive CAR materials sufficiently
close in time such that the inventive CAR materials can enhance the
effect of one or more additional therapeutic agents, or vice versa.
In this regard, the inventive CAR materials can be administered
first and the one or more additional therapeutic agents can be
administered second, or vice versa. Alternatively, the inventive
CAR materials and the one or more additional therapeutic agents can
be administered simultaneously.
[0124] An exemplary therapeutic agent that can be co-administered
with the CAR materials is a T cell active cytokine, such as IL-2.
It is believed that IL-2 enhances the therapeutic effect of the
inventive CAR materials. Without being bound by a particular theory
or mechanism, it is believed that IL-2 enhances therapy by
enhancing the in vivo expansion of the numbers and/or effector
function of cells expressing the inventive CARs. Other exemplary
cytokines include IL-7 and IL-15. For purposes of the inventive
methods, wherein host cells or populations of cells are
administered to the mammal, the cells can be cells that are
allogeneic or autologous to the mammal.
[0125] It is contemplated that the inventive CARs materials can be
used in methods of treating or preventing a disease in a mammal.
Without being bound to a particular theory or mechanism, the
inventive CARs have biological activity, e.g., ability to recognize
antigen, e.g., TSLPR, such that the CAR when expressed by a cell is
able to mediate an immune response against the cell expressing the
antigen, e.g., TSLPR, for which the CAR is specific. In this
regard, an embodiment of the invention provides a method of
treating or preventing cancer in a mammal, comprising administering
to the mammal the CARs, the nucleic acids, the recombinant
expression vectors, the host cells, the population of cells, the
antibodies and/or the antigen binding portions thereof, and/or the
pharmaceutical compositions of the invention in an amount effective
to treat or prevent cancer in the mammal.
[0126] An embodiment of the invention further comprises
lymphodepleting the mammal prior to administering the inventive CAR
materials. Examples of lymphodepletion include, but may not be
limited to, nonmyeloablative lymphodepleting chemotherapy,
myeloablative lymphodepleting chemotherapy, total body irradiation,
etc.
[0127] For purposes of the inventive methods, wherein host cells or
populations of cells are administered, the cells can be cells that
are allogeneic or autologous to the mammal. Preferably, the cells
are autologous to the mammal.
[0128] The mammal referred to herein can be any mammal. As used
herein, the term "mammal" refers to any mammal, including, but not
limited to, mammals of the order Rodentia, such as mice and
hamsters, and mammals of the order Logomorpha, such as rabbits. The
mammals may be from the order Carnivora, including Felines (cats)
and Canines (dogs). The mammals may be from the order Artiodactyla,
including Bovines (cows) and Swines (pigs) or of the order
Perssodactyla, including Equines (horses). The mammals may be of
the order Primates, Ceboids, or Simoids (monkeys) or of the order
Anthropoids (humans and apes). Preferably, the mammal is a
human.
[0129] With respect to the inventive methods, the cancer can be any
cancer, including any of acute lymphocytic cancer, acute myeloid
leukemia, alveolar rhabdomyosarcoma, bladder cancer (e.g., bladder
carcinoma), bone cancer, brain cancer (e.g., medulloblastoma),
breast cancer, cancer of the anus, anal canal, or anorectum, cancer
of the eye, cancer of the intrahepatic bile duct, cancer of the
joints, cancer of the neck, gallbladder, or pleura, cancer of the
nose, nasal cavity, or middle ear, cancer of the oral cavity,
cancer of the vulva, chronic lymphocytic leukemia, chronic myeloid
cancer, colon cancer, esophageal cancer, cervical cancer,
fibrosarcoma, gastrointestinal carcinoid tumor, head and neck
cancer (e.g., head and neck squamous cell carcinoma), Hodgkin
lymphoma, hypopharynx cancer, kidney cancer, larynx cancer,
leukemia, liquid tumors, liver cancer, lung cancer (e.g., non-small
cell lung carcinoma and lung adenocarcinoma), lymphoma,
mesothelioma, mastocytoma, melanoma, multiple myeloma, nasopharynx
cancer, non-Hodgkin lymphoma, B-chronic lymphocytic leukemia, hairy
cell leukemia, acute lymphocytic leukemia (ALL), and Burkitt's
lymphoma, ovarian cancer, pancreatic cancer, peritoneum, omentum,
and mesentery cancer, pharynx cancer, prostate cancer, rectal
cancer, renal cancer, skin cancer, small intestine cancer, soft
tissue cancer, solid tumors, synovial sarcoma, gastric cancer,
testicular cancer, thyroid cancer, and ureter cancer. Preferably,
the cancer is characterized by the expression of TSLPR.
[0130] The terms "treat," and "prevent" as well as words stemming
therefrom, as used herein, do not necessarily imply 100% or
complete treatment or prevention. Rather, there are varying degrees
of treatment or prevention of which one of ordinary skill in the
art recognizes as having a potential benefit or therapeutic effect.
In this respect, the inventive methods can provide any amount or
any level of treatment or prevention of cancer in a mammal.
Furthermore, the treatment or prevention provided by the inventive
method can include treatment or prevention of one or more
conditions or symptoms of the disease, e.g., cancer, being treated
or prevented. Also, for purposes herein, "prevention" can encompass
delaying the onset of the disease, or a symptom or condition
thereof.
[0131] Another embodiment of the invention provides a method of
detecting the presence of cancer in a mammal, comprising: (a)
contacting a sample comprising one or more cells from the mammal
with the CARs, the nucleic acids, the recombinant expression
vectors, the host cells, the population of cells, the antibodies,
and/or the antigen binding portions thereof, or the pharmaceutical
compositions of the invention, thereby forming a complex, (b) and
detecting the complex, wherein detection of the complex is
indicative of the presence of cancer in the mammal.
[0132] Another embodiment of the invention includes a method of
determining whether a subject with a proliferative disorder is a
candidate for treatment with a chimeric antigen receptor comprising
an antigen binding domain specific for TSLPR, the method comprising
measuring TSLPR expression levels in a biological sample from the
subject; and determining if the TSLPR expression levels of the
biological sample are increased compared to a sample from a control
subject without the proliferative disorder.
[0133] The sample may be obtained by any suitable method, e.g.,
biopsy or necropsy. A biopsy is the removal of tissue and/or cells
from an individual. Such removal may be to collect tissue and/or
cells from the individual in order to perform experimentation on
the removed tissue and/or cells. This experimentation may include
experiments to determine if the individual has and/or is suffering
from a certain condition or disease-state. The condition or disease
may be, e.g., cancer.
[0134] With respect to an embodiment of the inventive method of
detecting the presence of a proliferative disorder, e.g., cancer,
in a mammal, the sample comprising cells of the mammal can be a
sample comprising whole cells, lysates thereof, or a fraction of
the whole cell lysates, e.g., a nuclear or cytoplasmic fraction, a
whole protein fraction, or a nucleic acid fraction. If the sample
comprises whole cells, the cells can be any cells of the mammal,
e.g., the cells of any organ or tissue, including blood cells or
endothelial cells.
[0135] The contacting can take place in vitro or in vivo with
respect to the mammal. Preferably, the contacting is in vitro.
[0136] Also, detection of the complex can occur through any number
of ways known in the art. For instance, the inventive CARs,
polypeptides, proteins, nucleic acids, recombinant expression
vectors, host cells, populations of cells, or antibodies, or
antigen binding portions thereof, described herein, can be labeled
with a detectable label such as, for instance, a radioisotope, a
fluorophore (e.g., fluorescein isothiocyanate (FITC), phycoerythrin
(PE)), an enzyme (e.g., alkaline phosphatase, horseradish
peroxidase), and element particles (e.g., gold particles).
[0137] Methods of testing a CAR for the ability to recognize target
cells and for antigen specificity are known in the art. For
instance, Clay et al., J. Immunol., 163: 507-513 (1999), teaches
methods of measuring the release of cytokines (e.g.,
interferon-.gamma., granulocyte/monocyte colony stimulating factor
(GM-CSF), tumor necrosis factor a (TNF-.alpha.) or interleukin 2
(IL-2)). In addition, CAR function can be evaluated by measurement
of cellular cytotoxicity, as described in Zhao et al., J. Immunol.,
174: 4415-4423 (2005).
[0138] Another embodiment of the invention provides the use of the
CARs, nucleic acids, recombinant expression vectors, host cells,
populations of cells, antibodies, or antigen binding portions
thereof, and/or pharmaceutical compositions of the invention, for
the treatment or prevention of a proliferative disorder, e.g.,
cancer, in a mammal. The cancer may be any of the cancers described
herein. Preferably, the cancer is BCP-ALL.
[0139] The following examples further illustrate the invention but,
of course, should not be construed as in any way limiting its
scope.
Example 1
[0140] This example demonstrates the generation and testing of the
3G11 TSLPR CARs Short 3G11 (SEQ ID NOS: 39 and 43) and Long 3G11
(SEQ ID NOS: 40 and 44). The leader sequence is initially encoded
and enhances trafficking to the cell surface. It is likely to be
cleaved off in the mature form.
[0141] The following B cell acute lymphoblastic leukemia (ALL) cell
lines were used: MUTZ-5 (DSMZ ACC 490), REH-TSLPR (transduced with
human TSLPR) and REH as a TSLPR negative control. Cell line
cultures in media were supplemented with 10% heat-inactivated FBS
(Gemini Bioproducts, West Sacramento, Calif., USA), 10 mM HEPES,
100 U/mL penicillin, 100 ug/mL streptomycin, 2 mM L-glutamine
(Invitrogen, Carlsbad, Calif., USA). The 293T retroviral vector
packaging cell line (Clonetech, Mountain View, Calif., USA) was
cultured in DMEM (Invitrogen). In addition, pre-B cell ALL
xenografts JH331, JH352, NH362 which naturally overexpress TSLPR
were used as in vivo models. These are patient-derived ALL
xenografts established after patient consent on an IRB-approved
protocol. Human PBMCs from healthy donors were obtained from the
Department of Transfusion Medicine at the NIH Clinical Center,
under an NIH IRB approved protocol after informed consent in
accordance with the Declaration of Helsinki. The human PBMC were
cultured in AIMV with 5% FBS.
[0142] Construction of TSLPR chimeric antigen receptors. TSLPR
binding single chain fragment variable (scFv) sequences were
determined from the anti-TSLPR producing hybridoma 3G11 (Lu et al.,
J. Exp. Med., 2009, 206:2111-2119, incorporated herein by
reference). 3G11 was cultured in RPMI 1640 medium with sodium
pyruvate (1 mM), penicillin streptomycin (pen/strep) and 10% Fetal
Bovine serum. When the cells were ready to split, the medium was
changed to RPMI medium plus sodium pyruvate, pen/strep, and 5% of
ultra-low IgG FBS from GIBCO (Grand Island, N.Y., USA; Cat#16250)
for antibody production or harvesting the cells for total RNA
extraction. 3G11 total RNA were extracted with RNeasy Mini kit
(Qiagen, Valencia, Calif., USA) and then reverse transcribed into
cDNA with SuperScript III (Invitrogen). The cDNA were subsequently
used for PCR amplification with the combination of the degenerated
primers from the variable region of the heavy chain and the
constant gamma chain for the variable region of the heavy chain
(V.sub.H), and similarly, with the degenerated primer from the
kappa variable region and the specific primer from the kappa chain
constant region for the kappa light chain (V.sub.L) (Kettleborough
et al., Eur. J. Immunol., 1993, 23:206-211, incorporated by
reference herein in its entirety). The PCR reagents were either
purchased from Roche Diagnostics (PCR Buffer Set, Indianapolis,
Ind., USA) or from New England BioLabs (One Taq DNA Polymerase,
Ipswich, Mass., USA). The following PCR conditions were applied for
the amplification: 95.degree. C. for 1 min, 35 cycles of
(95.degree. C. for 15 sec, 50.degree. C. for 30 sec, 68.degree. C.
for 45 sec), final extension at 68.degree. C. for 5 min. The
resulting PCR products were gel purified and cloned into TOPO
vector (TOPO TA Cloning Kit for Sequencing, Invitrogen) and then
transformed into One Shot.RTM. TOP10 Chemically Competent E. coli
(Invitrogen). Single clones were picked for mini-prep and the
resulting plasmids were sent for sequencing analysis. To overcome
the secondary structure at the beginning of the heavy chain
variable region, a new antibody subtype specific reverse primer was
designed which is closer to the beginning of the 5' to combine with
the degenerated primer at the 5' end for amplification of the 5'
region of the V.sub.H. A PCR enhancer Betaine was used at 1 M to
facilitate the PCR reaction. For construction of the long CAR
constructs, the CH2CH3 domains from IGHG1 (gb|AAC82527.1 aa 98-329)
were included. The leader sequence for the scFv coding for T-cell
surface glycoprotein CD8 alpha chain was included to facilitate
membrane trafficking. The CAR-encoding amino acid sequences were
reverse translated, codon optimized, and synthesized as single
constructs (DNA 2.0, Menlo Park, Calif., USA). These constructs
were then subcloned into a third generation lentivirial plasmid
(pELNS-19BBzeta) containing a CD8 transmembrane domain, a 41BB
(CD137) signaling domain and a CD3zeta domain (previously described
in Hudecek et al., "The non-signaling extracellular spacer domain
of chimeric antigen receptors is decisive for in vivo antitumor
activity," Cancer Immunol. Res., 2014 and Milone et al., Molecular
Therapy, 2009, 17(8): 1453-1464, each of which are incorporated
herein by reference).
[0143] Lentiviral vector production and T cell transduction. TSLPR
CAR-encoding lentiviral vectors were produced by transient
transfection of the 293T cell line as previously described in
Hudecek et al., supra, and Milone et al., supra. Briefly, 293T
cells were plated into poly-D lysine coated 15 cm plates (BD
Bioscience, San Jose, Calif., USA). The following day, 293T cells
were transfected using lipofectamine 2000 (Invitrogen) with
plasmids encoding the TSLPR CAR along with packaging and envelope
vectors pMDLg/pRRE, pMD-G, and pRSV-Rev which were kindly provided
by Dr. R. Morgan (Surgery Branch, Center for Cancer Research, NCI,
NIH). Lenti-viral supernatants were collected 48 to 72 hours
post-transfection, centrifuged at 3000 RPM for 10 minutes to remove
cell debris, then stored at -80.degree. C. Human PBMCs from normal
donors were activated with a 1:1 ratio of CD3/CD28 microbeads
(Invitrogen) in AIM-V media containing 40 IU/mL recombinant IL-2
(teceleukin, rhIL-2; Roche, Indianapolis, Ind., USA) for 24 hours.
Activated T cells were resuspended at 2 million cells per 3 ml of
lentiviral supernatants plus 1 ml of fresh AIM-V media with 10
.mu.g/ml protamine sulfate and 40 IU/ml IL2 and cultured in 6-well
plates. Plates were centrifuged at 1000 g for 2 hours at 32.degree.
C. and then were incubated at 37.degree. C. overnight. A second
transduction was performed the following day. On the third day
following transduction, the CD3/CD28 beads were removed and the
cells were cultured at 300,000 cells/mL in AIM-V containing 100
IU/mL IL-2 with fresh IL2-containing media added every 2 to 3 days
until harvest at day 8 or 9.
[0144] Flow cytometry analysis. Surface expression of
CAR-transduced T cells was determined by flow cytometry using a
TSLPR-Fc (R&D Systems, Minneapolis, Minn., USA) followed by
incubation with PE-F(ab).sub.2 or APC-F(ab).sub.2 specific for
human IgG-Fc (Jackson ImmunoResearch Laboratories, West Grove, Pa.,
USA). Alternatively, biotin-conjugated protein L (Thermo
Scientific, Waltham, Mass., USA) was used to detect CAR expression
after incubation with streptavidin-conjugated PE (BD Bioscience).
Expression of CD19, CD22, and TSLPR on leukemia lines were detected
using the following anti-human antibodies: CD45-PerCP-Cy5.5
(eBioscience, San Diego, Calif., USA), CD19-Pac-Blue, CD19-APC-Cy7,
CD10_PE-Cy7, and CD22-PE, TSLPR-APC (BioLegend, San Diego, Calif.,
USA), and the T cells were characterized with the following
antibodies: CD3-APC-Cy7, CCR7-FITC (CD197), CD45RA-APC,
CD4-PacBlue, (BioLegend), CD45-PerCP-Cy5.5 (eBioscience), CD8-V500
(BD, Franklin Lakes, N.J., USA). The binding of the 3G11 hybridoma
supernatant to the TSLPR expression ALL lines was detected with
Goat-anti-mouse-PE (BD BioScience). Dead cells were excluded by
staining with Fixable Viability Dye eFluor.RTM. 506
(eBioscience).
[0145] Cellular cytotoxicity and cytokine assays. Both REH-TSLPR
and MUTZ5 cell lines express high level of TSLPR. REH was used as
negative control for TSLPR expression. Target cells were labeled
with 100 uCi .sup.51Cr (Perkin Elmer, Waltham, Mass., USA) for 1
hour. After washing, 5,000 targets per well were coincubated for 4
to 6 hours with bead-purified Pan T Cell II isolation kit (Miltenyi
Biotec, San Diego, Calif., USA) transduced T cells at various
effector to target (E:T) ratios. Assay supernatants were counted
for .sup.51Cr release using LumaPlates (Perkin Elmer) and a Top
Count Reader (Packard, Meriden, Conn.). Specific lysis was
calculated as follows: % Lysis=(experimental Lysis-spontaneous
lysis)/(maximum lysis-spontaneous lysis).times.100. Cytokine levels
in supernatants were determined after 24-hours using a multiplex
assay (Meso Scale Discovery, Rockville, Md., USA). For studies
including K562 cells. K562 cells are immortalized human myelogenous
erythroleukemia. They do not express TSLPR on the cell surface and
are normally used for detecting NK activity. K562 and REH were used
as negative target controls, the REH-TSLPR and MUTZ5 were used as
the positive target controls. The CAR transduced T cells were NK
depleted with Pan-T isolation kit and then incubated with the
different target cells. For cytokine production, the following
protocol was used: count the target cell and wash 3.times. times
and resuspend in RPMI at 1E6/ml, and put 100 ul into each well in
96-well plate (Final 1E5/well); count transduced T cell and wash
3.times. times and resuspend in RPMI at 1E6/ml, and put 100 ul into
each well in 96-well plate (final 1E5/well); set up a T cell only
and tumor cell only; incubate for 24 hours at 37.degree. C. and
harvest 100 ul of the supernatant for testing of the cytokines
production. All samples were in triplicate.
[0146] In vivo studies. Animal studies were carried out under
protocols approved by the NCI Bethesda Animal Care and Use
Committee. Pre B cell ALL cell lines and xenografts were IV
injected into NSG mice (NOD scid gamma, NOD.Cg-Prkdcscid Il2rgtm1
Wjl/SzJ JAX, Jackson ImmunoResearch Laboratories). For
luciferase-expressing lines, leukemia was detected using the
Xenogen IVIS Lumina (Caliper Life Sciences). NSG mice were injected
intraperitoneally with 3 mg D-luciferin (Caliper Life Sciences) and
were imaged 6 minutes later with an exposure time of 3 min. Living
Image Version 4.1 software (Caliper Life Sciences, Hopkinton,
Mass., USA) was used to analyze the bioluminescent signals for each
mouse as photons/s/cm.sup.2/sr. Non-luciferase expressing
xenografts were tracked with flow cytometry of peripheral blood or
bone marrow.
[0147] Binding of an anti-TSLPR antibody, produced by the 3G11
hybridoma, to TSLPR-overexpressing precursor-B cell acute
lymphoblastic leukemias ("TSLPRhi ALL") was confirmed (FIG. 1).
FIG. 2 shows expression determined by a commercial TSLPR antibody.
The sequences for the heavy and light chain variable regions (Fv)
were then determined. Single chain Fv (scFv) sequences were
constructed using a glycine linker and inserted into a chimeric
antigen receptor lentiviral vector backbone encoding CD8.alpha.
hinge and CD8 transmembrane regions with CD3zeta and 41BB (CD137)
intracellular domains (FIGS. 3 and 4). Because distance of the scFv
from the T cell surface may affect CAR function, a construct
containing an immunoglobulin CH2CH3 spacer domain between the scFv
and the transmembrane sequence was also generated for the "long"
CAR. Lentiviral vectors encoding the TSLPR CARs were then used to
transduce CD3/CD28 bead-activated human T cells resulting in a high
efficiency of gene transfer as detected by both protein L and a
TSLPR Fc fusion protein (FIGS. 5A and 5B). Although transduction
occurred on days 2 and 3 of culture, the fraction of CAR-expressing
T cells increased during subsequent culture, suggesting
preferential survival or enhanced expansion of T cells expressing
the CAR construct. TSLPR has limited expression in normal tissues
outside of the immune system. It has been found on dendritic cells
and subsets of activated T cells. Based on immunohistochemistry on
a normal pediatric tissue microarray, where there were scattered
rare cells in lymphoid tissues with robust membraneous expression,
possibly representing dendritic cells. There was also some staining
in pancreas, renal tubular cells, and colonic mucosa, where the
staining in these tissues was not consistent with cell surface
expression. There was no staining in the heart. As has been shown
with some primary preB ALL, a TSLPRhi ALL cell line (MUTZ5) and a
human TSLPRhi xenograph (JHH331) express TSLPR at comparable levels
to CD19 and CD22 (FIG. 2).
[0148] Testing was performed to determine whether T cells
transduced with the TSLPR CAR constructs demonstrate activity
against the pre B ALL cell line REH transduced to express TSLPR
(REH-TSLPR) as well as a naturally TSLPR over-expressing ALL line
(MUTZ5). As shown in FIG. 6, both short and long CAR T cells
produce high levels of interferon gamma (IFN.gamma.) and tumor
necrosis factor alpha (TNF.alpha.) when incubated with REH-TSLPR.
In addition, T cells with TSLPR CAR produce a broad range of
inflammatory cytokines in the presence of both TSLPR-transduced and
naturally overexpressing ALL cells (FIGS. 7A-7H and 8A-8E). When
the lytic capacity of TSLPR CAR T cells was measured, the short and
long constructs demonstrated equivalent activity against REH-TSLPR.
However, the short TSLPR CAR mediated greater lysis of MUTZ5 than
the long CAR despite comparable levels of TSLPR on both REH-TSLPR
and MUTZ5 (FIGS. 2 and 9A-D).
[0149] The ability for TSLPR CAR T cells to reduce ALL when infused
into mice bearing TSLPR-overexpressing ALL was tested next. Four
days after IV injection of luciferase-expressing REH-TSLPR,
leukemia was detectable at low levels. Injection of
15.times.10.sup.6 short CAR T cells appeared to completely reduce
ALL assessed by imaging (FIG. 10) and by flow cytometry of
peripheral blood for the presence of CD45+/GFP+ cells at day 12
following leukemia injection. (FIG. 11). Interestingly, despite
equivalent in vitro activity against REH-TSLPR, long CAR T cells
had minimal impact on leukemia progression in mice assessed by
imaging with some evidence for reduced leukemic burden in
peripheral blood when compared to mice receiving GFP-transduced T
cells (albeit not statistically different). To determine the reason
for the disparate activity of the long and short CAR constructs CAR
T cell persistence by flow cytometry were investigated, and it was
found that short CAR T cells were present in greater numbers in the
peripheral blood compared to long CAR T cells (FIGS. 12A-B). FIG.
12B shows TSLPR Fc v. CD8. CAR T v. CD45 showed the following
results: on Day 16, the short CART cells were at 3.02% (CD45 subset
of 5.36%) and the long CAR T cells were at 0.038% (CD45 subset
0.213%); on Day 27, the short CAR T cells were at 44.1% (CD45
subset of 89.4%) and the long CART cells were at 2.14% (CD45 subset
7.54%). Interestingly, the presence in greater numbers in the
peripheral blood of short CAR T cells was most notable at later
time points despite progression of ALL that maintained expression
of TSLPR in mice receiving long TSLPR CAR T cells. Thus, the marked
increase in activity seen with the short TSLPR CAR construct
compared to the long construct was associated with greater
persistence of short CAR-expressing T cells.
[0150] To test the short TSLPR CAR T cells in more established
leukemia, infusion was delayed until day 16 after REH-TSLPR
injection (FIG. 13). Remarkably, 10.times.10.sup.6 short TSLPR CAR
T cells were still able to induce rapid clearance of TSLPRhi ALL
that was maintained in the majority of mice through day 40 (FIG.
13). Importantly, there was no evidence for any alteration in the
progression of TSLPR when not over-expressed ("TSLPRlo ALL") by
TSLPR CAR T cells demonstrating that activity is dependent on
expression of the CAR target. Analysis of the relapses in mice
treated with the short TSLPR CAR T cells demonstrated retained
expression of CD19, CD10 and TSLPR indicating that failure is not
due to loss of antigen. CD8+ T cells generally exhibit greater in
vitro lytic function and are thought to be important mediators of
direct anti-tumor activity in vivo when compared to CD4+ T cells.
Interestingly, although the in vitro expansion protocol utilized in
these experiments resulted in a predominance of CD4+ T cells prior
to infusion, by day 50, CD8+ T cells expanded markedly and
represented the largest T cell subset in vivo (FIG. 14). This
expansion of CD8+ CAR-expressing T cells was associated with
expression of surface markers associated with effector phenotypes
by day 50 (FIG. 15, showing the physical distribution of the TSLPR
CAR). There were also a substantial percentage of CAR T cells with
a CCR7+/CD45RA phenotype, consistent with central memory subset,
thought to be important for persistence and sustained anti-tumor
activity.
[0151] An in vivo short CAR T cell dose titration was performed to
define the range over which activity is observed and whether short
TSLPR could reduce established leukemia. As shown in FIGS. 16A-C,
short TSLPR CAR cells at 15.times.10.sup.6 cells per mouse greatly
reduced ALL with clear activity at 5-10.times.10.sup.6 per mouse
and some activity as low as 1.times.10.sup.6 per mouse,
particularly noted as improved survival (FIG. 16C). Again, there
was minimal activity seen following infusion of the long CAR T
cells with only a slight decrease in leukemic burden in the
peripheral blood (FIG. 11) and luciferase activity at day 6 (FIG.
16B) compared to GFP T cells but no difference between the two
groups at any of the later time points consistent with the failure
of the long CAR T cells to persist.
[0152] The short TSLPR construct was tested in 3 xenograft models
of pre-B cell ALL that naturally overexpress TSLPR. As shown in
FIG. 17, the short TSLPR CAR greatly reduced human ALL TSLPRhi
xenograft expressing luciferase. The Short TSLPR CAR also greatly
reduced additional TSLPRhi xenografts from both the blood and bone
marrow of mice (FIGS. 18A and 18B).
[0153] FIG. 19 shows the short TSLPR CAR can reduce ALL in patient
xerographs with as low as 1.2 million of CART cells. FIG. 20 is a
dot plot showing the percentage of CAR T cells presented in mouse
blood sample. FIG. 21 shows the shift of the CD4 to CD8 of the CAR
T cells after injected in vivo.
[0154] The short TSLPR CAR was tested against an aggressive TSLPR
ALL that results in lethality by 60 days after IV injection in NSG
mice. One million aggressive TSLPRhi ALL cells were injected in NSG
mice intravenously on day 1 then treated with 1.2 million TSLPR
CAR+ T cells on day 14. The short TSLPR CAR demonstrated potent
activity (FIGS. 22-25), resulting in reduction in splenomegaly and
reduction in blast counts in the spleen and blood as early as day
14 following CAR injection. Interestingly, although there appeared
to be activity in the bone marrow as well, the clearance of
leukemia was less rapid and not statistically significant at this
early evaluation time. However, CAR treatment was associated with
eventual clearance of aggressive TSLPRhi ALL and prolonged
survival.
[0155] The activity of the short TSLPR CAR activity was compared to
that of a CD19 CAR containing the same scFv (FM68
scFv-CD8-CD137-CD3zeta) and a CD22 CAR construct (m971
scFv-CD8-CD137-CD3zeta). All had the same 41BB costimulatory domain
and resulted in comparable transduction efficiencies. The short
TSLPR CAR was comparable to both the CD19 and CD22 CARs at reducing
the JHH331 Luc TSLPRhi xenograph (FIG. 24).
Example 2
[0156] This example demonstrates the testing of two additional
TSLPR CARs (Short 2D10 (SEQ ID NOS: 41 and 45) and Long 2D10 (SEQ
ID NOS: 42 and 46)) and comparison to the CARs of Example 1. The
leader sequence is initially encoded and enhances trafficking to
the cell surface. It is likely to be cleaved off in the mature
form.
[0157] Methods of Example 1 were generally followed.
[0158] FIGS. 25A-C and 26 present the results. FIGS. 25A-C show the
cytolytic functions of the transduced T cells with different TSLPR
CAR constructs when incubated with TSLPR expression leukemia cell
lines, where the REH was served as a negative expression line. FIG.
26 shows therapeutic function of the different CAR constructs in
vivo.
[0159] All references, including publications, patent applications,
and patents, cited herein are hereby incorporated by reference to
the same extent as if each reference were individually and
specifically indicated to be incorporated by reference and were set
forth in its entirety herein.
[0160] The use of the terms "a" and "an" and "the" and "at least
one" and similar referents in the context of describing the
invention (especially in the context of the following claims) are
to be construed to cover both the singular and the plural, unless
otherwise indicated herein or clearly contradicted by context. The
use of the term "at least one" followed by a list of one or more
items (for example, "at least one of A and B") is to be construed
to mean one item selected from the listed items (A or B) or any
combination of two or more of the listed items (A and B), unless
otherwise indicated herein or clearly contradicted by context. The
terms "comprising," "having," "including," and "containing" are to
be construed as open-ended terms (i.e., meaning "including, but not
limited to,") unless otherwise noted. Also, everywhere "comprising"
(or its equivalent) is recited, the "comprising" is considered to
incorporate "consisting essentially of" and "consisting of." Thus,
an embodiment "comprising" (an) element(s) supports embodiments
"consisting essentially of" and "consisting of" the recited
element(s). Everywhere "consisting essentially of" is recited is
considered to incorporate "consisting of." Thus, an embodiment
"consisting essentially of" (an) element(s) supports embodiments
"consisting of" the recited element(s). Recitation of ranges of
values herein are merely intended to serve as a shorthand method of
referring individually to each separate value falling within the
range, unless otherwise indicated herein, and each separate value
is incorporated into the specification as if it were individually
recited herein. All methods described herein can be performed in
any suitable order unless otherwise indicated herein or otherwise
clearly contradicted by context. The use of any and all examples,
or exemplary language (e.g., "such as") provided herein, is
intended merely to better illuminate the invention and does not
pose a limitation on the scope of the invention unless otherwise
claimed. No language in the specification should be construed as
indicating any non-claimed element as essential to the practice of
the invention.
[0161] Preferred embodiments of this invention are described
herein, including the best mode known to the inventors for carrying
out the invention. Variations of those preferred embodiments may
become apparent to those of ordinary skill in the art upon reading
the foregoing description. The inventors expect skilled artisans to
employ such variations as appropriate, and the inventors intend for
the invention to be practiced otherwise than as specifically
described herein. Accordingly, this invention includes all
modifications and equivalents of the subject matter recited in the
claims appended hereto as permitted by applicable law. Moreover,
any combination of the above-described elements in all possible
variations thereof is encompassed by the invention unless otherwise
indicated herein or otherwise clearly contradicted by context.
Sequence CWU 1
1
461243PRTArtificial SequenceSynthetic 1Gln Val Thr Leu Lys Glu Ser
Gly Pro Gly Ile Leu Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr
Cys Ser Phe Ser Gly Phe Ser Leu Ser Thr Ser 20 25 30 Gly Met Gly
Val Gly Trp Ile Arg Gln Pro Ser Gly Lys Gly Leu Glu 35 40 45 Trp
Leu Ala His Ile Trp Trp Asp Asp Asp Lys Tyr Tyr Asn Pro Ser 50 55
60 Leu Lys Ser Gln Leu Thr Ile Ser Lys Asp Thr Ser Arg Asn Gln Val
65 70 75 80 Phe Leu Lys Ile Thr Ser Val Asp Thr Ala Asp Thr Ala Thr
Tyr Tyr 85 90 95 Cys Ser Arg Arg Pro Arg Gly Thr Met Asp Ala Met
Asp Tyr Trp Gly 100 105 110 Gln Gly Thr Ser Val Thr Val Ser Ser Gly
Gly Gly Gly Ser Gly Gly 115 120 125 Gly Gly Ser Gly Gly Gly Gly Ser
Asp Ile Val Met Thr Gln Ala Ala 130 135 140 Ser Ser Leu Ser Ala Ser
Leu Gly Asp Arg Val Thr Ile Ser Cys Arg 145 150 155 160 Ala Ser Gln
Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro 165 170 175 Asp
Gly Thr Val Lys Leu Leu Ile Tyr Tyr Thr Ser Arg Leu His Ser 180 185
190 Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser
195 200 205 Leu Thr Ile Arg Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr
Phe Cys 210 215 220 Gln Gln Val Tyr Thr Leu Pro Trp Thr Phe Gly Gly
Gly Thr Lys Leu 225 230 235 240 Glu Ile Lys 2245PRTArtificial
SequenceSynthetic 2Gln Val Thr Leu Lys Glu Ser Gly Pro Gly Ile Leu
Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr Cys Ser Phe Ser Gly
Phe Ser Leu Asn Thr Ser 20 25 30 Gly Met Gly Val Gly Trp Ile Arg
Gln Pro Ser Gly Lys Gly Leu Glu 35 40 45 Trp Leu Ala His Ile Trp
Trp Asp Asp Asp Lys Tyr Tyr Asn Pro Ser 50 55 60 Leu Lys Ser Gln
Leu Thr Ile Ser Lys Asp Thr Ser Arg Asn Gln Val 65 70 75 80 Phe Leu
Lys Ile Thr Ser Val Asp Thr Ala Asp Ser Ala Thr Tyr Tyr 85 90 95
Cys Ala Arg Arg Ala Ser His Val Ser Thr Val Asp Ser Phe Asp Phe 100
105 110 Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser Gly Gly Gly Gly
Ser 115 120 125 Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln
Met Thr Gln 130 135 140 Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly Asp
Arg Val Thr Ile Ser 145 150 155 160 Cys Arg Ala Ser Gln Asp Ile Ser
Asn Tyr Leu Asn Trp Phe Gln Gln 165 170 175 Lys Pro Asp Gly Thr Val
Lys Leu Leu Ile Tyr Tyr Thr Ser Arg Leu 180 185 190 His Ser Gly Val
Pro Ser Lys Phe Ser Gly Ser Gly Ser Gly Thr Asp 195 200 205 Tyr Ser
Leu Thr Ile Ser Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr 210 215 220
Phe Cys Gln Gln Gly Tyr Thr Leu Pro Trp Thr Phe Gly Gly Gly Thr 225
230 235 240 Lys Leu Glu Ile Lys 245 34PRTArtificial
SequenceSynthetic 3Ala Ser Ala Thr 1 41PRTArtificial
SequenceSynthetic 4Met 1 520PRTArtificial SequenceSynthetic 5Ala
Leu Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu His 1 5 10
15 Ala Ala Arg Pro 20 625PRTArtificial SequenceSynthetic 6Gln Val
Thr Leu Lys Glu Ser Gly Pro Gly Ile Leu Lys Pro Ser Gln 1 5 10 15
Thr Leu Ser Leu Thr Cys Ser Phe Ser 20 25 710PRTArtificial
SequenceSynthetic 7Gly Phe Ser Leu Ser Thr Ser Gly Met Gly 1 5 10
810PRTArtificial SequenceSynthetic 8Gly Phe Ser Leu Asn Thr Ser Gly
Met Gly 1 5 10 917PRTArtificial SequenceSynthetic 9Val Gly Trp Ile
Arg Gln Pro Ser Gly Lys Gly Leu Glu Trp Leu Ala 1 5 10 15 His
107PRTArtificial SequenceSynthetic 10Ile Trp Trp Asp Asp Asp Lys 1
5 1138PRTArtificial SequenceSynthetic 11Tyr Tyr Asn Pro Ser Leu Lys
Ser Gln Leu Thr Ile Ser Lys Asp Thr 1 5 10 15 Ser Arg Asn Gln Val
Phe Leu Lys Ile Thr Ser Val Asp Thr Ala Asp 20 25 30 Thr Ala Thr
Tyr Tyr Cys 35 1238PRTArtificial SequenceSynthetic 12Tyr Tyr Asn
Pro Ser Leu Lys Ser Gln Leu Thr Ile Ser Lys Asp Thr 1 5 10 15 Ser
Arg Asn Gln Val Phe Leu Lys Ile Thr Ser Val Asp Thr Ala Asp 20 25
30 Ser Ala Thr Tyr Tyr Cys 35 1313PRTArtificial SequenceSynthetic
13Ser Arg Arg Pro Arg Gly Thr Met Asp Ala Met Asp Tyr 1 5 10
1415PRTArtificial SequenceSynthetic 14Ala Arg Arg Ala Ser His Val
Ser Thr Val Asp Ser Phe Asp Phe 1 5 10 15 1511PRTArtificial
SequenceSynthetic 15Trp Gly Gln Gly Thr Ser Val Thr Val Ser Ser 1 5
10 1611PRTArtificial SequenceSynthetic 16Trp Gly Gln Gly Thr Thr
Leu Thr Val Ser Ser 1 5 10 1715PRTArtificial SequenceSynthetic
17Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser 1 5
10 15 1826PRTArtificial SequenceSynthetic 18Asp Ile Val Met Thr Gln
Ala Ala Ser Ser Leu Ser Ala Ser Leu Gly 1 5 10 15 Asp Arg Val Thr
Ile Ser Cys Arg Ala Ser 20 25 1926PRTArtificial SequenceSynthetic
19Asp Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly 1
5 10 15 Asp Arg Val Thr Ile Ser Cys Arg Ala Ser 20 25
206PRTArtificial SequenceSynthetic 20Gln Asp Ile Ser Lys Tyr 1 5
216PRTArtificial SequenceSynthetic 21Gln Asp Ile Ser Asn Tyr 1 5
2217PRTArtificial SequenceSynthetic 22Leu Asn Trp Tyr Gln Gln Lys
Pro Asp Gly Thr Val Lys Leu Leu Ile 1 5 10 15 Tyr 2317PRTArtificial
SequenceSynthetic 23Leu Asn Trp Phe Gln Gln Lys Pro Asp Gly Thr Val
Lys Leu Leu Ile 1 5 10 15 Tyr 243PRTArtificial SequenceSynthetic
24Tyr Thr Ser 1 2536PRTArtificial SequenceSynthetic 25Arg Leu His
Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly 1 5 10 15 Thr
Asp Tyr Ser Leu Thr Ile Arg Asn Leu Glu Gln Glu Asp Ile Ala 20 25
30 Thr Tyr Phe Cys 35 2636PRTArtificial SequenceSynthetic 26Arg Leu
His Ser Gly Val Pro Ser Lys Phe Ser Gly Ser Gly Ser Gly 1 5 10 15
Thr Asp Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln Glu Asp Ile Ala 20
25 30 Thr Tyr Phe Cys 35 279PRTArtificial SequenceSynthetic 27Gln
Gln Val Tyr Thr Leu Pro Trp Thr 1 5 289PRTArtificial
SequenceSynthetic 28Gln Gln Gly Tyr Thr Leu Pro Trp Thr 1 5
2910PRTArtificial SequenceSynthetic 29Phe Gly Gly Gly Thr Lys Leu
Glu Ile Lys 1 5 10 304PRTArtificial SequenceSynthetic 30Leu Glu Asp
Pro 1 31126PRTArtificial SequenceSynthetic 31Ala Glu Pro Lys Ser
Pro Asp Lys Thr His Thr Cys Pro Pro Cys Pro 1 5 10 15 Ala Pro Glu
Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys 20 25 30 Pro
Lys Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val 35 40
45 Val Val Asp Val Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr
50 55 60 Val Asp Gly Val Glu Val His Asn Ala Lys Thr Lys Pro Arg
Glu Glu 65 70 75 80 Gln Tyr Asn Ser Thr Tyr Arg Val Val Ser Val Leu
Thr Val Leu His 85 90 95 Gln Asp Trp Leu Asn Gly Lys Glu Tyr Lys
Cys Lys Val Ser Asn Lys 100 105 110 Ala Leu Pro Ala Pro Ile Glu Lys
Thr Ile Ser Lys Ala Lys 115 120 125 32107PRTArtificial
SequenceSynthetic 32Gly Gln Pro Arg Glu Pro Gln Val Tyr Thr Leu Pro
Pro Ser Arg Asp 1 5 10 15 Glu Leu Thr Lys Asn Gln Val Ser Leu Thr
Cys Leu Val Lys Gly Phe 20 25 30 Tyr Pro Ser Asp Ile Ala Val Glu
Trp Glu Ser Asn Gly Gln Pro Glu 35 40 45 Asn Asn Tyr Lys Thr Thr
Pro Pro Val Leu Asp Ser Asp Gly Ser Phe 50 55 60 Phe Leu Tyr Ser
Lys Leu Thr Val Asp Lys Ser Arg Trp Gln Gln Gly 65 70 75 80 Asn Val
Phe Ser Cys Ser Val Met His Glu Ala Leu His Asn His Tyr 85 90 95
Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly Lys 100 105
334PRTArtificial SequenceSynthetic 33Lys Asp Pro Lys 1
342PRTArtificial SequenceSynthetic 34Ser Gly 1 3545PRTArtificial
SequenceSynthetic 35Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro Ala
Pro Thr Ile Ala 1 5 10 15 Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala
Cys Arg Pro Ala Ala Gly 20 25 30 Gly Ala Val His Thr Arg Gly Leu
Asp Phe Ala Cys Asp 35 40 45 3624PRTArtificial SequenceSynthetic
36Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys Gly Val Leu Leu Leu 1
5 10 15 Ser Leu Val Ile Thr Leu Tyr Cys 20 3742PRTArtificial
SequenceSynthetic 37Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys
Gln Pro Phe Met 1 5 10 15 Arg Pro Val Gln Thr Thr Gln Glu Glu Asp
Gly Cys Ser Cys Arg Phe 20 25 30 Pro Glu Glu Glu Glu Gly Gly Cys
Glu Leu 35 40 38112PRTArtificial SequenceSynthetic 38Arg Val Lys
Phe Ser Arg Ser Ala Asp Ala Pro Ala Tyr Lys Gln Gly 1 5 10 15 Gln
Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr 20 25
30 Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys
35 40 45 Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu
Gln Lys 50 55 60 Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly Met
Lys Gly Glu Arg 65 70 75 80 Arg Arg Gly Lys Gly His Asp Gly Leu Tyr
Gln Gly Leu Ser Thr Ala 85 90 95 Thr Lys Asp Thr Tyr Asp Ala Leu
His Met Gln Ala Leu Pro Pro Arg 100 105 110 39489PRTArtificial
SequenceSynthetic 39Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu
Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Gln Val Thr Leu Lys
Glu Ser Gly Pro Gly Ile 20 25 30 Leu Lys Pro Ser Gln Thr Leu Ser
Leu Thr Cys Ser Phe Ser Gly Phe 35 40 45 Ser Leu Ser Thr Ser Gly
Met Gly Val Gly Trp Ile Arg Gln Pro Ser 50 55 60 Gly Lys Gly Leu
Glu Trp Leu Ala His Ile Trp Trp Asp Asp Asp Lys 65 70 75 80 Tyr Tyr
Asn Pro Ser Leu Lys Ser Gln Leu Thr Ile Ser Lys Asp Thr 85 90 95
Ser Arg Asn Gln Val Phe Leu Lys Ile Thr Ser Val Asp Thr Ala Asp 100
105 110 Thr Ala Thr Tyr Tyr Cys Ser Arg Arg Pro Arg Gly Thr Met Asp
Ala 115 120 125 Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser
Ser Gly Gly 130 135 140 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
Gly Ser Asp Ile Val 145 150 155 160 Met Thr Gln Ala Ala Ser Ser Leu
Ser Ala Ser Leu Gly Asp Arg Val 165 170 175 Thr Ile Ser Cys Arg Ala
Ser Gln Asp Ile Ser Lys Tyr Leu Asn Trp 180 185 190 Tyr Gln Gln Lys
Pro Asp Gly Thr Val Lys Leu Leu Ile Tyr Tyr Thr 195 200 205 Ser Arg
Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly Ser Gly Ser 210 215 220
Gly Thr Asp Tyr Ser Leu Thr Ile Arg Asn Leu Glu Gln Glu Asp Ile 225
230 235 240 Ala Thr Tyr Phe Cys Gln Gln Val Tyr Thr Leu Pro Trp Thr
Phe Gly 245 250 255 Gly Gly Thr Lys Leu Glu Ile Lys Ser Gly Thr Thr
Thr Pro Ala Pro 260 265 270 Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala
Ser Gln Pro Leu Ser Leu 275 280 285 Arg Pro Glu Ala Cys Arg Pro Ala
Ala Gly Gly Ala Val His Thr Arg 290 295 300 Gly Leu Asp Phe Ala Cys
Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly 305 310 315 320 Thr Cys Gly
Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys 325 330 335 Arg
Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg 340 345
350 Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro
355 360 365 Glu Glu Glu Glu Gly Gly Cys Glu Leu Arg Val Lys Phe Ser
Arg Ser 370 375 380 Ala Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln
Leu Tyr Asn Glu 385 390 395 400 Leu Asn Leu Gly Arg Arg Glu Glu Tyr
Asp Val Leu Asp Lys Arg Arg 405 410 415 Gly Arg Asp Pro Glu Met Gly
Gly Lys Pro Arg Arg Lys Asn Pro Gln 420 425 430 Glu Gly Leu Tyr Asn
Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr 435 440 445 Ser Glu Ile
Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp 450 455 460 Gly
Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala 465 470
475 480 Leu His Met Gln Ala Leu Pro Pro Arg 485 40730PRTArtificial
SequenceSynthetic 40Met Ala Leu Pro Val Thr Ala Leu Leu Leu Pro Leu
Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg Pro Gln Val Thr Leu Lys
Glu Ser Gly Pro Gly Ile 20 25 30 Leu Lys Pro Ser Gln Thr Leu Ser
Leu Thr Cys Ser Phe Ser Gly Phe 35 40 45 Ser Leu Ser Thr Ser Gly
Met Gly Val Gly Trp Ile Arg Gln Pro Ser 50 55 60 Gly Lys Gly Leu
Glu Trp Leu Ala His Ile Trp Trp Asp Asp Asp Lys 65 70 75 80 Tyr Tyr
Asn Pro Ser Leu Lys Ser Gln Leu Thr Ile Ser Lys Asp Thr 85 90 95
Ser Arg Asn Gln Val Phe Leu Lys Ile Thr Ser Val Asp Thr Ala Asp 100
105 110 Thr Ala Thr Tyr Tyr Cys Ser Arg Arg Pro Arg Gly Thr Met Asp
Ala 115 120 125 Met Asp Tyr Trp Gly Gln Gly Thr Ser Val Thr Val Ser
Ser Gly Gly 130 135 140 Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly
Gly Ser Asp Ile Val 145 150 155 160 Met Thr Gln Ala Ala Ser Ser Leu
Ser Ala Ser Leu Gly Asp Arg Val 165 170 175 Thr Ile Ser Cys Arg Ala
Ser Gln Asp Ile Ser Lys Tyr Leu Asn Trp 180 185 190 Tyr Gln Gln Lys
Pro Asp Gly Thr Val Lys Leu Leu Ile Tyr Tyr Thr 195 200 205 Ser Arg
Leu His Ser Gly Val Pro Ser Arg Phe Ser Gly Ser
Gly Ser 210 215 220 Gly Thr Asp Tyr Ser Leu Thr Ile Arg Asn Leu Glu
Gln Glu Asp Ile 225 230 235 240 Ala Thr Tyr Phe Cys Gln Gln Val Tyr
Thr Leu Pro Trp Thr Phe Gly 245 250 255 Gly Gly Thr Lys Leu Glu Ile
Lys Leu Glu Asp Pro Ala Glu Pro Lys 260 265 270 Ser Pro Asp Lys Thr
His Thr Cys Pro Pro Cys Pro Ala Pro Glu Leu 275 280 285 Leu Gly Gly
Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr 290 295 300 Leu
Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val 305 310
315 320 Ser His Glu Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly
Val 325 330 335 Glu Val His Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln
Tyr Asn Ser 340 345 350 Thr Tyr Arg Val Val Ser Val Leu Thr Val Leu
His Gln Asp Trp Leu 355 360 365 Asn Gly Lys Glu Tyr Lys Cys Lys Val
Ser Asn Lys Ala Leu Pro Ala 370 375 380 Pro Ile Glu Lys Thr Ile Ser
Lys Ala Lys Gly Gln Pro Arg Glu Pro 385 390 395 400 Gln Val Tyr Thr
Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys Asn Gln 405 410 415 Val Ser
Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala 420 425 430
Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr 435
440 445 Pro Pro Val Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys
Leu 450 455 460 Thr Val Asp Lys Ser Arg Trp Gln Gln Gly Asn Val Phe
Ser Cys Ser 465 470 475 480 Val Met His Glu Ala Leu His Asn His Tyr
Thr Gln Lys Ser Leu Ser 485 490 495 Leu Ser Pro Gly Lys Lys Asp Pro
Lys Ser Gly Thr Thr Thr Pro Ala 500 505 510 Pro Arg Pro Pro Thr Pro
Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser 515 520 525 Leu Arg Pro Glu
Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr 530 535 540 Arg Gly
Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala 545 550 555
560 Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys
565 570 575 Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro
Phe Met 580 585 590 Arg Pro Val Gln Thr Thr Gln Glu Glu Asp Gly Cys
Ser Cys Arg Phe 595 600 605 Pro Glu Glu Glu Glu Gly Gly Cys Glu Leu
Arg Val Lys Phe Ser Arg 610 615 620 Ser Ala Asp Ala Pro Ala Tyr Lys
Gln Gly Gln Asn Gln Leu Tyr Asn 625 630 635 640 Glu Leu Asn Leu Gly
Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg 645 650 655 Arg Gly Arg
Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro 660 665 670 Gln
Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala 675 680
685 Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His
690 695 700 Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr
Tyr Asp 705 710 715 720 Ala Leu His Met Gln Ala Leu Pro Pro Arg 725
730 41491PRTArtificial SequenceSynthetic 41Met Ala Leu Pro Val Thr
Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His Ala Ala Arg
Pro Gln Val Thr Leu Lys Glu Ser Gly Pro Gly Ile 20 25 30 Leu Lys
Pro Ser Gln Thr Leu Ser Leu Thr Cys Ser Phe Ser Gly Phe 35 40 45
Ser Leu Asn Thr Ser Gly Met Gly Val Gly Trp Ile Arg Gln Pro Ser 50
55 60 Gly Lys Gly Leu Glu Trp Leu Ala His Ile Trp Trp Asp Asp Asp
Lys 65 70 75 80 Tyr Tyr Asn Pro Ser Leu Lys Ser Gln Leu Thr Ile Ser
Lys Asp Thr 85 90 95 Ser Arg Asn Gln Val Phe Leu Lys Ile Thr Ser
Val Asp Thr Ala Asp 100 105 110 Ser Ala Thr Tyr Tyr Cys Ala Arg Arg
Ala Ser His Val Ser Thr Val 115 120 125 Asp Ser Phe Asp Phe Trp Gly
Gln Gly Thr Thr Leu Thr Val Ser Ser 130 135 140 Gly Gly Gly Gly Ser
Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp 145 150 155 160 Ile Gln
Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly Asp 165 170 175
Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr Leu 180
185 190 Asn Trp Phe Gln Gln Lys Pro Asp Gly Thr Val Lys Leu Leu Ile
Tyr 195 200 205 Tyr Thr Ser Arg Leu His Ser Gly Val Pro Ser Lys Phe
Ser Gly Ser 210 215 220 Gly Ser Gly Thr Asp Tyr Ser Leu Thr Ile Ser
Asn Leu Glu Gln Glu 225 230 235 240 Asp Ile Ala Thr Tyr Phe Cys Gln
Gln Gly Tyr Thr Leu Pro Trp Thr 245 250 255 Phe Gly Gly Gly Thr Lys
Leu Glu Ile Lys Ser Gly Thr Thr Thr Pro 260 265 270 Ala Pro Arg Pro
Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu 275 280 285 Ser Leu
Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His 290 295 300
Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu 305
310 315 320 Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu Val Ile Thr
Leu Tyr 325 330 335 Cys Lys Arg Gly Arg Lys Lys Leu Leu Tyr Ile Phe
Lys Gln Pro Phe 340 345 350 Met Arg Pro Val Gln Thr Thr Gln Glu Glu
Asp Gly Cys Ser Cys Arg 355 360 365 Phe Pro Glu Glu Glu Glu Gly Gly
Cys Glu Leu Arg Val Lys Phe Ser 370 375 380 Arg Ser Ala Asp Ala Pro
Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr 385 390 395 400 Asn Glu Leu
Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys 405 410 415 Arg
Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn 420 425
430 Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu
435 440 445 Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg Arg Arg Gly
Lys Gly 450 455 460 His Asp Gly Leu Tyr Gln Gly Leu Ser Thr Ala Thr
Lys Asp Thr Tyr 465 470 475 480 Asp Ala Leu His Met Gln Ala Leu Pro
Pro Arg 485 490 42732PRTArtificial SequenceSynthetic 42Met Ala Leu
Pro Val Thr Ala Leu Leu Leu Pro Leu Ala Leu Leu Leu 1 5 10 15 His
Ala Ala Arg Pro Gln Val Thr Leu Lys Glu Ser Gly Pro Gly Ile 20 25
30 Leu Lys Pro Ser Gln Thr Leu Ser Leu Thr Cys Ser Phe Ser Gly Phe
35 40 45 Ser Leu Asn Thr Ser Gly Met Gly Val Gly Trp Ile Arg Gln
Pro Ser 50 55 60 Gly Lys Gly Leu Glu Trp Leu Ala His Ile Trp Trp
Asp Asp Asp Lys 65 70 75 80 Tyr Tyr Asn Pro Ser Leu Lys Ser Gln Leu
Thr Ile Ser Lys Asp Thr 85 90 95 Ser Arg Asn Gln Val Phe Leu Lys
Ile Thr Ser Val Asp Thr Ala Asp 100 105 110 Ser Ala Thr Tyr Tyr Cys
Ala Arg Arg Ala Ser His Val Ser Thr Val 115 120 125 Asp Ser Phe Asp
Phe Trp Gly Gln Gly Thr Thr Leu Thr Val Ser Ser 130 135 140 Gly Gly
Gly Gly Ser Gly Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp 145 150 155
160 Ile Gln Met Thr Gln Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly Asp
165 170 175 Arg Val Thr Ile Ser Cys Arg Ala Ser Gln Asp Ile Ser Asn
Tyr Leu 180 185 190 Asn Trp Phe Gln Gln Lys Pro Asp Gly Thr Val Lys
Leu Leu Ile Tyr 195 200 205 Tyr Thr Ser Arg Leu His Ser Gly Val Pro
Ser Lys Phe Ser Gly Ser 210 215 220 Gly Ser Gly Thr Asp Tyr Ser Leu
Thr Ile Ser Asn Leu Glu Gln Glu 225 230 235 240 Asp Ile Ala Thr Tyr
Phe Cys Gln Gln Gly Tyr Thr Leu Pro Trp Thr 245 250 255 Phe Gly Gly
Gly Thr Lys Leu Glu Ile Lys Leu Glu Asp Pro Ala Glu 260 265 270 Pro
Lys Ser Pro Asp Lys Thr His Thr Cys Pro Pro Cys Pro Ala Pro 275 280
285 Glu Leu Leu Gly Gly Pro Ser Val Phe Leu Phe Pro Pro Lys Pro Lys
290 295 300 Asp Thr Leu Met Ile Ser Arg Thr Pro Glu Val Thr Cys Val
Val Val 305 310 315 320 Asp Val Ser His Glu Asp Pro Glu Val Lys Phe
Asn Trp Tyr Val Asp 325 330 335 Gly Val Glu Val His Asn Ala Lys Thr
Lys Pro Arg Glu Glu Gln Tyr 340 345 350 Asn Ser Thr Tyr Arg Val Val
Ser Val Leu Thr Val Leu His Gln Asp 355 360 365 Trp Leu Asn Gly Lys
Glu Tyr Lys Cys Lys Val Ser Asn Lys Ala Leu 370 375 380 Pro Ala Pro
Ile Glu Lys Thr Ile Ser Lys Ala Lys Gly Gln Pro Arg 385 390 395 400
Glu Pro Gln Val Tyr Thr Leu Pro Pro Ser Arg Asp Glu Leu Thr Lys 405
410 415 Asn Gln Val Ser Leu Thr Cys Leu Val Lys Gly Phe Tyr Pro Ser
Asp 420 425 430 Ile Ala Val Glu Trp Glu Ser Asn Gly Gln Pro Glu Asn
Asn Tyr Lys 435 440 445 Thr Thr Pro Pro Val Leu Asp Ser Asp Gly Ser
Phe Phe Leu Tyr Ser 450 455 460 Lys Leu Thr Val Asp Lys Ser Arg Trp
Gln Gln Gly Asn Val Phe Ser 465 470 475 480 Cys Ser Val Met His Glu
Ala Leu His Asn His Tyr Thr Gln Lys Ser 485 490 495 Leu Ser Leu Ser
Pro Gly Lys Lys Asp Pro Lys Ser Gly Thr Thr Thr 500 505 510 Pro Ala
Pro Arg Pro Pro Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro 515 520 525
Leu Ser Leu Arg Pro Glu Ala Cys Arg Pro Ala Ala Gly Gly Ala Val 530
535 540 His Thr Arg Gly Leu Asp Phe Ala Cys Asp Ile Tyr Ile Trp Ala
Pro 545 550 555 560 Leu Ala Gly Thr Cys Gly Val Leu Leu Leu Ser Leu
Val Ile Thr Leu 565 570 575 Tyr Cys Lys Arg Gly Arg Lys Lys Leu Leu
Tyr Ile Phe Lys Gln Pro 580 585 590 Phe Met Arg Pro Val Gln Thr Thr
Gln Glu Glu Asp Gly Cys Ser Cys 595 600 605 Arg Phe Pro Glu Glu Glu
Glu Gly Gly Cys Glu Leu Arg Val Lys Phe 610 615 620 Ser Arg Ser Ala
Asp Ala Pro Ala Tyr Lys Gln Gly Gln Asn Gln Leu 625 630 635 640 Tyr
Asn Glu Leu Asn Leu Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp 645 650
655 Lys Arg Arg Gly Arg Asp Pro Glu Met Gly Gly Lys Pro Arg Arg Lys
660 665 670 Asn Pro Gln Glu Gly Leu Tyr Asn Glu Leu Gln Lys Asp Lys
Met Ala 675 680 685 Glu Ala Tyr Ser Glu Ile Gly Met Lys Gly Glu Arg
Arg Arg Gly Lys 690 695 700 Gly His Asp Gly Leu Tyr Gln Gly Leu Ser
Thr Ala Thr Lys Asp Thr 705 710 715 720 Tyr Asp Ala Leu His Met Gln
Ala Leu Pro Pro Arg 725 730 43468PRTArtificial SequenceSynthetic
43Gln Val Thr Leu Lys Glu Ser Gly Pro Gly Ile Leu Lys Pro Ser Gln 1
5 10 15 Thr Leu Ser Leu Thr Cys Ser Phe Ser Gly Phe Ser Leu Ser Thr
Ser 20 25 30 Gly Met Gly Val Gly Trp Ile Arg Gln Pro Ser Gly Lys
Gly Leu Glu 35 40 45 Trp Leu Ala His Ile Trp Trp Asp Asp Asp Lys
Tyr Tyr Asn Pro Ser 50 55 60 Leu Lys Ser Gln Leu Thr Ile Ser Lys
Asp Thr Ser Arg Asn Gln Val 65 70 75 80 Phe Leu Lys Ile Thr Ser Val
Asp Thr Ala Asp Thr Ala Thr Tyr Tyr 85 90 95 Cys Ser Arg Arg Pro
Arg Gly Thr Met Asp Ala Met Asp Tyr Trp Gly 100 105 110 Gln Gly Thr
Ser Val Thr Val Ser Ser Gly Gly Gly Gly Ser Gly Gly 115 120 125 Gly
Gly Ser Gly Gly Gly Gly Ser Asp Ile Val Met Thr Gln Ala Ala 130 135
140 Ser Ser Leu Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser Cys Arg
145 150 155 160 Ala Ser Gln Asp Ile Ser Lys Tyr Leu Asn Trp Tyr Gln
Gln Lys Pro 165 170 175 Asp Gly Thr Val Lys Leu Leu Ile Tyr Tyr Thr
Ser Arg Leu His Ser 180 185 190 Gly Val Pro Ser Arg Phe Ser Gly Ser
Gly Ser Gly Thr Asp Tyr Ser 195 200 205 Leu Thr Ile Arg Asn Leu Glu
Gln Glu Asp Ile Ala Thr Tyr Phe Cys 210 215 220 Gln Gln Val Tyr Thr
Leu Pro Trp Thr Phe Gly Gly Gly Thr Lys Leu 225 230 235 240 Glu Ile
Lys Ser Gly Thr Thr Thr Pro Ala Pro Arg Pro Pro Thr Pro 245 250 255
Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu Ala Cys 260
265 270 Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu Asp Phe
Ala 275 280 285 Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys
Gly Val Leu 290 295 300 Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys
Arg Gly Arg Lys Lys 305 310 315 320 Leu Leu Tyr Ile Phe Lys Gln Pro
Phe Met Arg Pro Val Gln Thr Thr 325 330 335 Gln Glu Glu Asp Gly Cys
Ser Cys Arg Phe Pro Glu Glu Glu Glu Gly 340 345 350 Gly Cys Glu Leu
Arg Val Lys Phe Ser Arg Ser Ala Asp Ala Pro Ala 355 360 365 Tyr Lys
Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu Gly Arg 370 375 380
Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp Pro Glu 385
390 395 400 Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly Leu
Tyr Asn 405 410 415 Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser
Glu Ile Gly Met 420 425 430 Lys Gly Glu Arg Arg Arg Gly Lys Gly His
Asp Gly Leu Tyr Gln Gly 435 440 445 Leu Ser Thr Ala Thr Lys Asp Thr
Tyr Asp Ala Leu His Met Gln Ala 450 455 460 Leu Pro Pro Arg 465
44709PRTArtificial SequenceSynthetic 44Gln Val Thr Leu Lys Glu Ser
Gly Pro Gly Ile Leu Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser Leu Thr
Cys Ser Phe Ser Gly Phe Ser Leu Ser Thr Ser 20 25 30 Gly Met Gly
Val Gly Trp Ile Arg Gln Pro Ser Gly Lys Gly Leu Glu 35 40 45 Trp
Leu Ala His Ile Trp Trp Asp Asp Asp Lys Tyr Tyr Asn Pro Ser 50 55
60 Leu Lys Ser Gln Leu Thr Ile Ser Lys Asp Thr Ser Arg Asn Gln Val
65 70 75
80 Phe Leu Lys Ile Thr Ser Val Asp Thr Ala Asp Thr Ala Thr Tyr Tyr
85 90 95 Cys Ser Arg Arg Pro Arg Gly Thr Met Asp Ala Met Asp Tyr
Trp Gly 100 105 110 Gln Gly Thr Ser Val Thr Val Ser Ser Gly Gly Gly
Gly Ser Gly Gly 115 120 125 Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile
Val Met Thr Gln Ala Ala 130 135 140 Ser Ser Leu Ser Ala Ser Leu Gly
Asp Arg Val Thr Ile Ser Cys Arg 145 150 155 160 Ala Ser Gln Asp Ile
Ser Lys Tyr Leu Asn Trp Tyr Gln Gln Lys Pro 165 170 175 Asp Gly Thr
Val Lys Leu Leu Ile Tyr Tyr Thr Ser Arg Leu His Ser 180 185 190 Gly
Val Pro Ser Arg Phe Ser Gly Ser Gly Ser Gly Thr Asp Tyr Ser 195 200
205 Leu Thr Ile Arg Asn Leu Glu Gln Glu Asp Ile Ala Thr Tyr Phe Cys
210 215 220 Gln Gln Val Tyr Thr Leu Pro Trp Thr Phe Gly Gly Gly Thr
Lys Leu 225 230 235 240 Glu Ile Lys Leu Glu Asp Pro Ala Glu Pro Lys
Ser Pro Asp Lys Thr 245 250 255 His Thr Cys Pro Pro Cys Pro Ala Pro
Glu Leu Leu Gly Gly Pro Ser 260 265 270 Val Phe Leu Phe Pro Pro Lys
Pro Lys Asp Thr Leu Met Ile Ser Arg 275 280 285 Thr Pro Glu Val Thr
Cys Val Val Val Asp Val Ser His Glu Asp Pro 290 295 300 Glu Val Lys
Phe Asn Trp Tyr Val Asp Gly Val Glu Val His Asn Ala 305 310 315 320
Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser Thr Tyr Arg Val Val 325
330 335 Ser Val Leu Thr Val Leu His Gln Asp Trp Leu Asn Gly Lys Glu
Tyr 340 345 350 Lys Cys Lys Val Ser Asn Lys Ala Leu Pro Ala Pro Ile
Glu Lys Thr 355 360 365 Ile Ser Lys Ala Lys Gly Gln Pro Arg Glu Pro
Gln Val Tyr Thr Leu 370 375 380 Pro Pro Ser Arg Asp Glu Leu Thr Lys
Asn Gln Val Ser Leu Thr Cys 385 390 395 400 Leu Val Lys Gly Phe Tyr
Pro Ser Asp Ile Ala Val Glu Trp Glu Ser 405 410 415 Asn Gly Gln Pro
Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val Leu Asp 420 425 430 Ser Asp
Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val Asp Lys Ser 435 440 445
Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser Val Met His Glu Ala 450
455 460 Leu His Asn His Tyr Thr Gln Lys Ser Leu Ser Leu Ser Pro Gly
Lys 465 470 475 480 Lys Asp Pro Lys Ser Gly Thr Thr Thr Pro Ala Pro
Arg Pro Pro Thr 485 490 495 Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu
Ser Leu Arg Pro Glu Ala 500 505 510 Cys Arg Pro Ala Ala Gly Gly Ala
Val His Thr Arg Gly Leu Asp Phe 515 520 525 Ala Cys Asp Ile Tyr Ile
Trp Ala Pro Leu Ala Gly Thr Cys Gly Val 530 535 540 Leu Leu Leu Ser
Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly Arg Lys 545 550 555 560 Lys
Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg Pro Val Gln Thr 565 570
575 Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu Glu
580 585 590 Gly Gly Cys Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp
Ala Pro 595 600 605 Ala Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu
Leu Asn Leu Gly 610 615 620 Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys
Arg Arg Gly Arg Asp Pro 625 630 635 640 Glu Met Gly Gly Lys Pro Arg
Arg Lys Asn Pro Gln Glu Gly Leu Tyr 645 650 655 Asn Glu Leu Gln Lys
Asp Lys Met Ala Glu Ala Tyr Ser Glu Ile Gly 660 665 670 Met Lys Gly
Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu Tyr Gln 675 680 685 Gly
Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala Leu His Met Gln 690 695
700 Ala Leu Pro Pro Arg 705 45470PRTArtificial SequenceSynthetic
45Gln Val Thr Leu Lys Glu Ser Gly Pro Gly Ile Leu Lys Pro Ser Gln 1
5 10 15 Thr Leu Ser Leu Thr Cys Ser Phe Ser Gly Phe Ser Leu Asn Thr
Ser 20 25 30 Gly Met Gly Val Gly Trp Ile Arg Gln Pro Ser Gly Lys
Gly Leu Glu 35 40 45 Trp Leu Ala His Ile Trp Trp Asp Asp Asp Lys
Tyr Tyr Asn Pro Ser 50 55 60 Leu Lys Ser Gln Leu Thr Ile Ser Lys
Asp Thr Ser Arg Asn Gln Val 65 70 75 80 Phe Leu Lys Ile Thr Ser Val
Asp Thr Ala Asp Ser Ala Thr Tyr Tyr 85 90 95 Cys Ala Arg Arg Ala
Ser His Val Ser Thr Val Asp Ser Phe Asp Phe 100 105 110 Trp Gly Gln
Gly Thr Thr Leu Thr Val Ser Ser Gly Gly Gly Gly Ser 115 120 125 Gly
Gly Gly Gly Ser Gly Gly Gly Gly Ser Asp Ile Gln Met Thr Gln 130 135
140 Thr Thr Ser Ser Leu Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser
145 150 155 160 Cys Arg Ala Ser Gln Asp Ile Ser Asn Tyr Leu Asn Trp
Phe Gln Gln 165 170 175 Lys Pro Asp Gly Thr Val Lys Leu Leu Ile Tyr
Tyr Thr Ser Arg Leu 180 185 190 His Ser Gly Val Pro Ser Lys Phe Ser
Gly Ser Gly Ser Gly Thr Asp 195 200 205 Tyr Ser Leu Thr Ile Ser Asn
Leu Glu Gln Glu Asp Ile Ala Thr Tyr 210 215 220 Phe Cys Gln Gln Gly
Tyr Thr Leu Pro Trp Thr Phe Gly Gly Gly Thr 225 230 235 240 Lys Leu
Glu Ile Lys Ser Gly Thr Thr Thr Pro Ala Pro Arg Pro Pro 245 250 255
Thr Pro Ala Pro Thr Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro Glu 260
265 270 Ala Cys Arg Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu
Asp 275 280 285 Phe Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly
Thr Cys Gly 290 295 300 Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr
Cys Lys Arg Gly Arg 305 310 315 320 Lys Lys Leu Leu Tyr Ile Phe Lys
Gln Pro Phe Met Arg Pro Val Gln 325 330 335 Thr Thr Gln Glu Glu Asp
Gly Cys Ser Cys Arg Phe Pro Glu Glu Glu 340 345 350 Glu Gly Gly Cys
Glu Leu Arg Val Lys Phe Ser Arg Ser Ala Asp Ala 355 360 365 Pro Ala
Tyr Lys Gln Gly Gln Asn Gln Leu Tyr Asn Glu Leu Asn Leu 370 375 380
Gly Arg Arg Glu Glu Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg Asp 385
390 395 400 Pro Glu Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu
Gly Leu 405 410 415 Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala
Tyr Ser Glu Ile 420 425 430 Gly Met Lys Gly Glu Arg Arg Arg Gly Lys
Gly His Asp Gly Leu Tyr 435 440 445 Gln Gly Leu Ser Thr Ala Thr Lys
Asp Thr Tyr Asp Ala Leu His Met 450 455 460 Gln Ala Leu Pro Pro Arg
465 470 46711PRTArtificial SequenceSynthetic 46Gln Val Thr Leu Lys
Glu Ser Gly Pro Gly Ile Leu Lys Pro Ser Gln 1 5 10 15 Thr Leu Ser
Leu Thr Cys Ser Phe Ser Gly Phe Ser Leu Asn Thr Ser 20 25 30 Gly
Met Gly Val Gly Trp Ile Arg Gln Pro Ser Gly Lys Gly Leu Glu 35 40
45 Trp Leu Ala His Ile Trp Trp Asp Asp Asp Lys Tyr Tyr Asn Pro Ser
50 55 60 Leu Lys Ser Gln Leu Thr Ile Ser Lys Asp Thr Ser Arg Asn
Gln Val 65 70 75 80 Phe Leu Lys Ile Thr Ser Val Asp Thr Ala Asp Ser
Ala Thr Tyr Tyr 85 90 95 Cys Ala Arg Arg Ala Ser His Val Ser Thr
Val Asp Ser Phe Asp Phe 100 105 110 Trp Gly Gln Gly Thr Thr Leu Thr
Val Ser Ser Gly Gly Gly Gly Ser 115 120 125 Gly Gly Gly Gly Ser Gly
Gly Gly Gly Ser Asp Ile Gln Met Thr Gln 130 135 140 Thr Thr Ser Ser
Leu Ser Ala Ser Leu Gly Asp Arg Val Thr Ile Ser 145 150 155 160 Cys
Arg Ala Ser Gln Asp Ile Ser Asn Tyr Leu Asn Trp Phe Gln Gln 165 170
175 Lys Pro Asp Gly Thr Val Lys Leu Leu Ile Tyr Tyr Thr Ser Arg Leu
180 185 190 His Ser Gly Val Pro Ser Lys Phe Ser Gly Ser Gly Ser Gly
Thr Asp 195 200 205 Tyr Ser Leu Thr Ile Ser Asn Leu Glu Gln Glu Asp
Ile Ala Thr Tyr 210 215 220 Phe Cys Gln Gln Gly Tyr Thr Leu Pro Trp
Thr Phe Gly Gly Gly Thr 225 230 235 240 Lys Leu Glu Ile Lys Leu Glu
Asp Pro Ala Glu Pro Lys Ser Pro Asp 245 250 255 Lys Thr His Thr Cys
Pro Pro Cys Pro Ala Pro Glu Leu Leu Gly Gly 260 265 270 Pro Ser Val
Phe Leu Phe Pro Pro Lys Pro Lys Asp Thr Leu Met Ile 275 280 285 Ser
Arg Thr Pro Glu Val Thr Cys Val Val Val Asp Val Ser His Glu 290 295
300 Asp Pro Glu Val Lys Phe Asn Trp Tyr Val Asp Gly Val Glu Val His
305 310 315 320 Asn Ala Lys Thr Lys Pro Arg Glu Glu Gln Tyr Asn Ser
Thr Tyr Arg 325 330 335 Val Val Ser Val Leu Thr Val Leu His Gln Asp
Trp Leu Asn Gly Lys 340 345 350 Glu Tyr Lys Cys Lys Val Ser Asn Lys
Ala Leu Pro Ala Pro Ile Glu 355 360 365 Lys Thr Ile Ser Lys Ala Lys
Gly Gln Pro Arg Glu Pro Gln Val Tyr 370 375 380 Thr Leu Pro Pro Ser
Arg Asp Glu Leu Thr Lys Asn Gln Val Ser Leu 385 390 395 400 Thr Cys
Leu Val Lys Gly Phe Tyr Pro Ser Asp Ile Ala Val Glu Trp 405 410 415
Glu Ser Asn Gly Gln Pro Glu Asn Asn Tyr Lys Thr Thr Pro Pro Val 420
425 430 Leu Asp Ser Asp Gly Ser Phe Phe Leu Tyr Ser Lys Leu Thr Val
Asp 435 440 445 Lys Ser Arg Trp Gln Gln Gly Asn Val Phe Ser Cys Ser
Val Met His 450 455 460 Glu Ala Leu His Asn His Tyr Thr Gln Lys Ser
Leu Ser Leu Ser Pro 465 470 475 480 Gly Lys Lys Asp Pro Lys Ser Gly
Thr Thr Thr Pro Ala Pro Arg Pro 485 490 495 Pro Thr Pro Ala Pro Thr
Ile Ala Ser Gln Pro Leu Ser Leu Arg Pro 500 505 510 Glu Ala Cys Arg
Pro Ala Ala Gly Gly Ala Val His Thr Arg Gly Leu 515 520 525 Asp Phe
Ala Cys Asp Ile Tyr Ile Trp Ala Pro Leu Ala Gly Thr Cys 530 535 540
Gly Val Leu Leu Leu Ser Leu Val Ile Thr Leu Tyr Cys Lys Arg Gly 545
550 555 560 Arg Lys Lys Leu Leu Tyr Ile Phe Lys Gln Pro Phe Met Arg
Pro Val 565 570 575 Gln Thr Thr Gln Glu Glu Asp Gly Cys Ser Cys Arg
Phe Pro Glu Glu 580 585 590 Glu Glu Gly Gly Cys Glu Leu Arg Val Lys
Phe Ser Arg Ser Ala Asp 595 600 605 Ala Pro Ala Tyr Lys Gln Gly Gln
Asn Gln Leu Tyr Asn Glu Leu Asn 610 615 620 Leu Gly Arg Arg Glu Glu
Tyr Asp Val Leu Asp Lys Arg Arg Gly Arg 625 630 635 640 Asp Pro Glu
Met Gly Gly Lys Pro Arg Arg Lys Asn Pro Gln Glu Gly 645 650 655 Leu
Tyr Asn Glu Leu Gln Lys Asp Lys Met Ala Glu Ala Tyr Ser Glu 660 665
670 Ile Gly Met Lys Gly Glu Arg Arg Arg Gly Lys Gly His Asp Gly Leu
675 680 685 Tyr Gln Gly Leu Ser Thr Ala Thr Lys Asp Thr Tyr Asp Ala
Leu His 690 695 700 Met Gln Ala Leu Pro Pro Arg 705 710
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